Protein kinase stress-related proteins and methods of use in plants

ABSTRACT

A transgenic plant transformed by a protein kinase stress-related protein (PKSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated PKSRP, and isolated nucleic acid coding PKSRP, and vectors and host cells containing the latter. Further provided are methods of producing transgenic plants expressing PKSRP, and methods of identifying novel PKSRP and methods of modifying the expression of PKSRP in plants.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to nucleic acid sequencesencoding proteins that are associated with abiotic stress responses andabiotic stress tolerance in plants. In particular, this inventionrelates to nucleic acid sequences encoding proteins that confer drought,cold, and/or salt tolerance to plants.

[0003] 2. Background Art

[0004] Environmental stress due to salinity and drought are among themost serious factors limiting the productivity of agricultural crops. Itis estimated that 35-45% of the 279 million hectares of land irrigationis presently affected by high salinity. This is exclusive of the regionsclassified as arid and desert lands. The consequence represents asignificant economic and political factor and contributes to foodshortages in many underdeveloped countries. In addition to salinitystress, crop yield losses due to drought in crops such as soybean, corn,rice and cotton also represent a significant economic factor. Moreover,drought is also responsible for food shortages in many countriesworldwide. Developing crops tolerant to salt and drought is a strategythat has potential to alleviate some of these adverse situations.

[0005] Traditional plant breeding strategies to develop new lines ofplants that exhibit tolerance to drought or salt tolerance arerelatively slow and require specific tolerant lines for crossing withthe desired commercial lines. Limited germplasm resources andincompatibility in crosses between distantly related plant species alsorepresent a significant problem encountered in conventional breeding. Incontrast, plant genetic transformation and availability of useful genessubjected to specific expression patterns allow one to generate stresstolerant plants using transgenic approaches.

[0006] Drought, cold as well as salt stresses have a common themeimportant for plant growth and that is water availability. Plants areexposed during their entire life cycle to conditions of reducedenvironmental water content. Most plants have evolved strategies toprotect themselves against these conditions of desiccation. However, ifthe severity and duration of the drought conditions are too great, theeffects on plant development, growth and yield of most crop plants areprofound. Since high salt content in some soils result in less availablewater for cell intake, its effect is similar to those observed underdrought conditions. Additionally, under freezing temperatures, plantcells loose water as a result of ice formation that starts in theapoplast and withdraws water from the symplast. Commonly, a plant'smolecular response mechanisms to each of these stress condition arecommon and protein kinases play an essential role in these molecularmechanisms.

[0007] Protein kinases represent a super family and the members of thisfamily catalyze the reversible transfer of a phosphate group of ATP toserine, threonine and tyrosine amino acid side chains on targetproteins. Protein kinases are primary elements in signaling processes inplants and have been reported to play crucial roles in perception andtransduction of signals that allow a cell (and the plant) to respond toenvironmental stimuli. In particular, receptor protein kinases (RPKs)represent one group of protein kinases that activate a complex array ofintracellular signaling pathways in response to the extracellularenvironment (Van der Gear et al., 1994 Annu. Rev. Cell Biol.10:251-337). RPKs are single-pass transmembrane proteins that contain anamino-terminal signal sequence, extracellular domains unique to eachreceptor, and a cytoplasmic kinase domain. Ligand binding induces homo-or hetero-dimerization of RPKs, and the resultant close proximity of thecytoplasmic domains results in kinase activation bytransphosphorylation. Although plants have many proteins similar toRPKs, no ligand has been identified for these receptor-like kinases(RLKs). The majority of plant RLKs that have been identified belong tothe family of Serine/Threonine (Ser/Thr) kinases, and most haveextracellular Leucine-rich repeats (Becraft, P W., 1998 Trends PlantSci. 3:384-388).

[0008] Another type of protein kinase is the Ca+-dependent proteinkinase (CDPK). This type of kinase has a calmodulin-like domain at theCOOH terminus which allows response to Ca+ signals directly withoutcalmodulin being present. Currently, CDPKs are the most prevalentSer/Thr protein kinases found in higher plants. Although theirphysiological roles remain unclear, they are induced by cold, droughtand abscisic acid (ABA) (Knight et al., 1991 Nature 352:524; Schroeder,J I and Thuleau, P., 1991 Plant Cell 3:555; Bush, D. S., 1995 Annu. Rev.Plant Phys. Plant Mol. Biol. 46:95; Urao, T. et al., 1994 Mol. Gen.Genet. 244:331).

[0009] Another type of signaling mechanism involves members of theconserved SNF1 Serine/Threonine protein kinase family. These kinasesplay essential roles in eukaryotic glucose and stress signaling (1).Plant SNF1-like kinases participate in the control of key metabolicenzymes, including HMGR, nitrate reductase, sucrose synthase, andsucrose phosphate synthase (SPS) (4). Genetic and biochemical dataindicate that sugar-dependent regulation of SNF1 kinases involvesseveral other sensory and signaling components in yeast, plants andanimals.

[0010] Additionally, members of the Mitogen-activated protein kinase(MAPK) family have been implicated in the actions of numerousenvironmental stresses in animals, yeasts and plants. It has beendemonstrated that both MAPK-like kinase activity and mRNA levels of thecomponents of MAPK cascades increase in response to environmental stressand plant hormone signal transduction. MAP kinases are components ofsequential kinase cascades, which are activated by phosphorylation ofthreonine and tyrosine residues by intermediate upstream MAP kinasekinases (MAPKKs). The MAPKKs are themselves activated by phosphorylationof serine and threonine residues by upstream kinases (MAPKKKs). A numberof MAP Kinase genes have been reported in higher plants.

SUMMARY OF THE INVENTION

[0011] The present invention provides a transgenic plant transformed bya protein kinase stress-related protein (PKSRP) coding nucleic acid,wherein expression of the nucleic acid sequence in the plant results inincreased tolerance to environmental stress as compared to a wild typevariety of the plant. The invention provides that the PKSRP can beselected from any of the well known general classes of protein kinaseproteins, including but not limited to: 1) Receptor Protein Kinases(RPK); 2) Receptor-Like Kinases (RLK); 3) Calcium Dependent ProteinKinases (CDPK); 4) SNF1 Serine/threonine Protein Kinases (SNF1); 5)Mitogen-activated Protein Kinases (MAPK); 6) intermediate upstreamMitogen-activated Protein Kinases (MAPKK); and upstreamMitogen-activated Protein Kinases (MAPKKK). The invention furtherprovides specific examples of PKSRP, and PKSRP coding nucleic acids,such as 1) PK-1; 2) PK-2; and 3) MPK-1.

[0012] The invention provides in some embodiments that the PKSRP andcoding nucleic acid are that found in members of the genusPhyscomitrella. In another preferred embodiment, the nucleic acid andprotein are from a Physcomitrella patens plant. The invention providesthat the environmental stress can be salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof In preferred embodiments, the environmental stress can besalinity and drought, or a combination thereof.

[0013] The invention further provides a seed produced by a transgenicplant transformed by a PKSRP coding nucleic acid, wherein the plant istrue breeding for increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention furtherprovides a seed produced by a transgenic plant expressing a PKSRP,wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.

[0014] The invention further provides an agricultural product producedby any of the transgenic plants described herein. The invention furtherprovides an isolated PKSRP, wherein the PKSRP is as described below. Theinvention further provides an isolated PKSRP coding nucleic acid,wherein the PKSRP coding nucleic acid codes for a PKSRP as describedbelow.

[0015] The invention further provides an isolated recombinant expressionvector comprising a nucleic acid as described below, wherein expressionof the vector in a host cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. The invention further provides a host cell containing the vectorand a plant containing the host cell.

[0016] The invention further provides a method of producing a transgenicplant with a PKSRP coding nucleic acid, wherein expression of thenucleic acid in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a PKSRP coding nucleic acid, and (b) generating from theplant cell a transgenic plant with an increased tolerance toenvironmental stress as compared to a wild type variety of the plant. Inpreferred embodiments, the PKSRP is as described below. In preferredembodiments, the PKSRP coding nucleic acid is as described below.

[0017] The present invention further provides a method of identifying anovel PKSRP, comprising (a) raising a specific antibody response to aPKSRP, or fragment thereof, as described above; (b) screening putativePKSRP material with the antibody, wherein specific binding of theantibody to the material indicates the presence of a potentially novelPKSRP; and (c) analyzing the bound material in comparison to known PKSRPto determine its novelty. Alternatively, hybridization with nucleic acidprobes as described below can be used to identify novel PKSRP nucleicacids.

[0018] The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a PKSRP inthe plant, wherein the PKSRP is as described below. The inventionprovides that this method can be performed such that the stresstolerance is either increased or decreased.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1(A-C) shows a nucleotide sequence of the partial PK-1 (SEQID NO: 1); PK-2 (SEQ ID NO: 2) and MPK-1 (SEQ ID NO: 3) fromPhyscomitrella patens

[0020]FIG. 2(A-C) shows a nucleotide sequence of the full-length PK-1(SEQ ID NO: 4), PK-2 (SEQ ID NO: 5) and MPK-1 (SEQ ID NO: 6) fromPhyscomitrella patens.

[0021]FIG. 3(A-C) shows a deduced amino acid sequence of PK-1 (SEQ IDNO: 7), PK-2 (SEQ ID NO: 8) and MPK-1 (SEQ ID NO: 9).

[0022]FIG. 4 shows a diagram of the plant expression vector pGMSGcontaining the Super promoter driving the expression of SEQ ID NOs:4, 5and 6. The components are: aacCI gentamycin resistance gene(Hajdukiewicz et al., 1994 Plant Molecular Biology 25:989-94), NOSpromoter (Becker et al., 1992 Plant Molecular Biology 20:1195-7), g7Tterminator (Becker et al., 1992), NOSpA terminator (Jefferson et al.,1987 EMBO J 6:3901-7). “Desired Gene” refers to the gene to beover-expressed in plants.

[0023]FIG. 5 shows the results of a drought stress test withover-expressing MPK-1 transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0024]FIG. 6 shows the results of a salt stress test withover-expressing MPK-1 transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0025]FIG. 7 shows the results of a drought stress test withover-expressing PK-2 transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0026]FIG. 8 shows the results of a Salt stress test withover-expressing PK-2 transgenic plants and wild-type Arabidopsis lines.The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included herein. However, before thepresent compounds, compositions, and methods are disclosed anddescribed, it is to be understood that this invention is not limited tospecific nucleic acids, specific polypeptides, specific cell types,specific host cells, specific conditions, or specific methods, etc., assuch may, of course, vary, and the numerous modifications and variationstherein will be apparent to those skilled in the art. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing specific embodiments only and is not intended to be limiting.In particular, the designation of the amino acid sequences as “ProteinKinase Stress-related Proteins” (PKSRPs), in no way limits thefunctionality of those sequences.

[0028] The present invention provides a transgenic plant transformed bya PKSRP coding nucleic acid, wherein expression of the nucleic acidsequence in the plant results in increased tolerance to environmentalstress as compared to a wild type variety of the plant. The inventionfurther provides a seed produced by a transgenic plant transformed by aPKSRP coding nucleic acid, wherein the seed contains the PKSRP codingnucleic acid, and wherein the plant is true breeding for increasedtolerance to environmental stress as compared to a wild type variety ofthe plant. The invention further provides a seed produced by atransgenic plant expressing a PKSRP, wherein the seed contains thePKSRP, and wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The invention further provides an agricultural product produced by anyof the above-or below-described transgenic plants. As used herein, theterm “variety” refers to a group of plants within a species that shareconstant characters that separate them from the typical form and fromother possible varieties within that species. While possessing at leastone distinctive trait, a variety is also characterized by some variationbetween individuals within the variety, based primarily on the Mendeliansegregation of traits among the progeny of succeeding generations. Avariety is considered “true breeding” for a particular trait if it isgenetically homozygous for that trait to the extent that, when thetrue-breeding variety is self-pollinated, a significant amount ofindependent segregation of the trait among the progeny is not observed.In the present invention, the trait arises from the transgenicexpression of a single DNA sequence introduced into a plant variety.

[0029] The invention further provides an isolated PKSRP. The inventionprovides that the PKSRP can be selected from one of the well knowngeneral classes of protein kinase proteins: 1) Receptor Protein Kinases(RPK); 2) Receptor-Like Kinases (RLK); 3) Calcium Dependent ProteinKinases (CDPK); 4) SNF1 Serine/threonine Protein Kinases (SNF1); 5)Mitogen-activated Protein Kinases (MAPK); 6) intermediate upstreamMitogen-activated Protein Kinases (MAPKK); and upstreamMitogen-activated Protein Kinases (MAPKKK). In further preferredembodiments, the PKSRP is selected from 1) Protein Kinase-1 (PK-1) asdefined in SEQ ID NO: 7; 2) Protein Kinase-1 (PK-2) as defined in SEQ IDNO: 8; and 3) Mitogen-activated Protein Kinase-1 (MPK-1) as defined inSEQ ID NO: 9; and homologues thereof. Homologues of the amino acidsequences are defined below.

[0030] The invention further provides an isolated PKSRP coding nucleicacid. In preferred embodiments the PKSRP coding nucleic acid is selectedfrom 1) Protein Kinase-1 (PK-1) as defined in SEQ ID NO: 4; 2) ProteinKinase-1 (PK-2) as defined in SEQ ID NO: 5; and 3) Mitogen-activatedProtein Kinase-1 (MPK-1) as defined in SEQ ID NO: 6; and homologuesthereof. Homologues of the nucleotide sequences are defined below. Thepresent invention includes PKSRP coding nucleic acids that encode PKSRPsas described herein. In some embodiments, the invention provides thatthe PKSRP is selected from 1) Protein Kinase-1 (PK-1) as defined in SEQID NO: 7; 2) Protein Kinase-1 (PK-2) as defined in SEQ ID NO: 8; and 3)Mitogen-activated Protein Kinase-1 (MPK-1) as defined in SEQ ID NO: 9.In one preferred embodiment, the nucleic acid and protein are isolatedfrom the plant genus Physcomitrella. In another preferred embodiment,the nucleic acid and protein are from a Physcomitrella patens (P.patens) plant.

[0031] As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can besalinity, drought, and temperature, or combinations thereof, and inparticular, can be high salinity, low water content and low temperature.It is also to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized.

[0032] In accordance with the purposes of this invention, as embodiedand broadly described herein, this invention, in one aspect, provides anisolated nucleic acid from a moss encoding a Stress-related Protein(SRP), or a portion thereof. In particular, the present inventionprovides nucleic acids encoding PKSRPs including the nucleic acidsequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Thepresent invention also provides amino acid sequences of PKSRPs includingthe amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ IDNO: 9. As mentioned above, the present invention describes for the firsttime the predicted P. patens proteins Protein Kinase-1 (PK-1), ProteinKinase-1 (PK-2) and Mitogen-activated Protein Kinase-1 (MPK-1). Thepresent invention also describes for the first time that the P. patensproteins Protein Kinase-1 (PK-1), Protein Kinase-1 (PK-2) andMitogen-activated Protein Kinase-1 (MPK-1), are useful for increasingstress tolerance in plants.

[0033] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5or SEQ ID NO: 6, or a portion thereof, can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, a P. patens PKSRP cDNA can be isolated from a P.patens library using all or portion of one of the sequences of SEQ IDNO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 as a hybridization probe andstandard hybridization techniques (e.g., as described in Sambrook etal., 1989 Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Moreover, a nucleic acid molecule encompassing all or aportion of one of the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ IDNO: 3 can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon this sequence (e.g., anucleic acid molecule encompassing all or a portion of one of thesequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 can be isolatedby the polymerase chain reaction using oligonucleotide primers designedbased upon this same sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ IDNO: 3). For example, mRNA can be isolated from plant cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979Biochemistry 18:5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney M L V reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the nucleotide sequences shown in SEQ IDNO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. A nucleic acid molecule of theinvention can be amplified using cDNA or, alternatively, genomic DNA, asa template and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid molecule so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a PKSRPnucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

[0034] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises one of the nucleotide sequences shown in SEQ IDNO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. The sequences of SEQ ID NO: 4, SEQID NO: 5 or SEQ ID NO: 6 correspond to the Physcomitrella patens PKSRPcDNAs of the invention. These cDNAs comprise sequences encoding PKSRPs(i.e., the “coding region”, indicated in Table 1), as well as 5′untranslated sequences and 3′ untranslated sequences. It is therefore tobe understood that SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 compriseboth coding regions and 5′ and 3′ untranslated regions. Alternatively,the nucleic acid molecule can comprise only the coding region of any ofthe sequences in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or cancontain whole genomic fragments isolated from genomic DNA. A codingregion of these sequences is indicated as “ORF position”.

[0035] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleic acid molecule which is acomplement of one of the nucleotide sequences shown in SEQ ID NO: 4, SEQID NO: 5 or SEQ ID NO: 6, or a portion thereof. A nucleic acid moleculewhich is complementary to one of the nucleotide sequences shown in SEQID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 is one which is sufficientlycomplementary to one of the nucleotide sequences shown in SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO: 6 such that it can hybridize to one of thenucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:6, thereby forming a stable duplex.

[0036] In still another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleotide sequence which is atleast about 50-60%, preferably at least about 60-70%, more preferably atleast about 70-80%, 80-90%, or 90-95%, and even more preferably at leastabout 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotidesequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or aportion thereof. In an additional preferred embodiment, an isolatednucleic acid molecule of the invention comprises a nucleotide sequencewhich hybridizes, e.g., hybridizes under stringent conditions, to one ofthe nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ IDNO: 6, or a portion thereof. These hybridization conditions includewashing with a solution having a salt concentration of about 0.02 molarat pH 7 at about 60° C.

[0037] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the coding region of one of the sequences in SEQ IDNO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, for example a fragment which canbe used as a probe or primer or a fragment encoding a biologicallyactive portion of a PKSRP. The nucleotide sequences determined from thecloning of the PKSRP genes from P. patens allows for the generation ofprobes and primers designed for use in identifying and/or cloning PKSRPhomologues in other cell types and organisms, as well as PKSRPhomologues from other mosses or related species. Therefore thisinvention also provides compounds comprising the nucleic acid moleculesdisclosed herein, or fragments thereof. These compounds include thenucleic acid molecules attached to a moiety. These moieties include, butare not limited to, detection moieties, hybridization moieties,purification moieties, delivery moieties, reaction moieties, bindingmoieties, and the like. The probe/primer typically comprisessubstantially isolated oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, preferably about 25, morepreferably about 40, 50 or 75 consecutive nucleotides of a sense strandof one of the sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQID NO: 6, an anti-sense sequence of one of the sequences set forth inSEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or naturally occurringmutants thereof. Primers based on a nucleotide sequence of SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO: 6 can be used in PCR reactions to clone PKSRPhomologues. Probes based on the PKSRP nucleotide sequences can be usedto detect transcripts or genomic sequences encoding the same orhomologous proteins. In preferred embodiments, the probe furthercomprises a label group attached thereto, e.g. the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a genomic marker test kit foridentifying cells which express an PKSRP, such as by measuring a levelof a PKSRP-encoding nucleic acid in a sample of cells, e.g., detectingPKSRP mRNA levels or determining whether a genomic PKSRP gene has beenmutated or deleted.

[0038] In particular, a useful method to ascertain the level oftranscription of the gene (an indicator of the amount of mRNA availablefor translation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: New York), in which a primer designed to bindto the gene of interest is labeled with a detectable tag (usuallyradioactive or chemiluminescent), such that when the total RNA of aculture of the organism is extracted, run on gel, transferred to astable matrix and incubated with this probe, the binding and quantity ofbinding of the probe indicates the presence and also the quantity ofmRNA for this gene. This information at least partially demonstrates thedegree of transcription of the transformed gene. Total cellular RNA canbe prepared from cells, tissues or organs by several methods, allwell-known in the art, such as that described in Bormann, E. R. et al.,1992 Mol. Microbiol. 6:317-326.

[0039] To assess the presence or relative quantity of protein translatedfrom this mRNA, standard techniques, such as a Western blot, may beemployed (see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: New York). In this process, total cellularproteins are extracted, separated by gel electrophoresis, transferred toa matrix such as nitrocellulose, and incubated with a probe, such as anantibody, which specifically binds to the desired protein. This probe isgenerally tagged with a chemiluminescent or calorimetric label that maybe readily detected. The presence and quantity of label observedindicates the presence and quantity of the desired mutant proteinpresent in the cell.

[0040] In one embodiment, the nucleic acid molecule of the inventionencodes a protein or portion thereof which includes an amino acidsequence which is sufficiently homologous to an amino acid sequence ofSEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 such that the protein orportion thereof maintains the same or a similar function as the aminoacid sequence to which it is compared. As used herein, the language“sufficiently homologous” refers to proteins or portions thereof whichhave amino acid sequences which include a minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chainas an amino acid residue in one of the ORFs of a sequence of SEQ ID NO:7, SEQ ID NO: 8 or SEQ ID NO: 9) amino acid residues to a PKSRP aminoacid sequence such that the protein or portion thereof is able toparticipate in a stress tolerance response in a plant, or moreparticularly can participate in protein kinase signal transductionmechanisms involved in a stress tolerance response in a Physcomitrellapatens plant. Examples of such activities are also described herein.Examples of PKSRP activities are set forth in Table 1.

[0041] In another embodiment, the protein is at least about 50-60%,preferably at least about 60-70%, and more preferably at least about70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%,98%, 99% or more homologous to an entire amino acid sequence shown inSEQ ID NO: 7; SEQ ID NO: 8 or SEQ ID NO: 9. In yet another embodiment,at least about 50-60%, preferably at least about 60-70%, and morepreferably at least about 70-80%, 80-90%, 90-95%, and most preferably atleast about 96%, 97%, 98%, 99% or more homologous to an entire aminoacid sequence encoded by a nucleic acid sequence shown in SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO: 6.

[0042] Portions of proteins encoded by the PKSRP nucleic acid moleculesof the invention are preferably biologically active portions of one ofthe PKSRPs. As used herein, the term “biologically active portion of aPKSRP” is intended to include a portion, e.g., a domain/motif, of aPKSRP that participates in a stress tolerance response in a plant, ormore particularly participates in the protein kinase signal transductionmechanisms involved in a stress tolerance response in a plant, or has anactivity as set forth in Table 1. To determine whether a PKSRP or abiologically active portion thereof can participate in protein kinasesignal transduction mechanisms involved in a stress tolerance responsein a plant, a stress analysis of a plant expressing the PKSRP may beperformed. Such analysis methods are well known to those skilled in theart, as detailed in Example 7.

[0043] Additional nucleic acid fragments encoding biologically activeportions of a PKSRP can be prepared by isolating a portion of one of thesequences in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, expressing theencoded portion of the PKSRP or peptide (e.g., by recombinant expressionin vitro) and assessing the activity of the encoded portion of the PKSRPor peptide.

[0044] The invention further encompasses nucleic acid molecules thatdiffer from one of the nucleotide sequences shown in SEQ ID NO: 4, SEQID NO: 5 or SEQ ID NO: 6 (and portions thereof) due to degeneracy of thegenetic code and thus encode the same PKSRP as that encoded by thenucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:6. In a further embodiment, the nucleic acid molecule of the inventionencodes a full length Physcomitrella patens protein which issubstantially homologous to an amino acid sequence of a polypeptideshown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0045] In addition to the Physcomitrella patens PKSRP nucleotidesequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, it willbe appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of PKSRPsmay exist within a population (e.g., the Physcomitrella patenspopulation). Such genetic polymorphism in the PKSRP gene may exist amongindividuals within a population due to natural variation. As usedherein, the terms “gene” and “recombinant gene” refer to nucleic acidmolecules comprising an open reading frame encoding a PKSRP, preferablya Physcomitrella patens PKSRP. Such natural variations can typicallyresult in 1-5% variance in the nucleotide sequence of the PKSRP gene.Any and all such nucleotide variations and resulting amino acidpolymorphisms in a PKSRP that are the result of natural variation andthat do not alter the functional activity of the PKSRPs are intended tobe within the scope of the invention.

[0046] Nucleic acid molecules corresponding to natural variants andnon-Physcomitrella patens homologues of the Physcomitrella patens PKSRPcDNA of the invention can be isolated based on their homology toPhyscomitrella patens PKSRP nucleic acid disclosed herein using thePhyscomitrella patens cDNA, or a portion thereof, as a hybridizationprobe according to standard hybridization techniques under stringenthybridization conditions. Accordingly, in another embodiment, anisolated nucleic acid molecule of the invention is at least 15nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO: 6. In other embodiments, the nucleic acid isat least 30, 50, 100, 250 or more nucleotides in length. As used herein,the term “hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 65%, more preferably at least about 70%, andeven more preferably at least about 75% or more homologous to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, 6.3.1-6.3.6, John Wiley & Sons, N.Y. (1989). Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2×SSC., 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to a sequence of SEQ ID NO: 4, SEQID NO: 5 or SEQ ID NO: 6 corresponds to a naturally occurring nucleicacid molecule. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein). In oneembodiment, the nucleic acid encodes a natural Physcomitrella patensPKSRP.

[0047] In addition to naturally-occurring variants of the PKSRP sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced by mutation into a nucleotidesequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, thereby leadingto changes in the amino acid sequence of the encoded PKSRP, withoutaltering the functional ability of the PKSRP. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in a sequence of SEQ ID NO: 4, SEQ IDNO: 5 or SEQ ID NO: 6. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of one of the PKSRPs(SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9) without altering theactivity of said PKSRP, whereas an “essential” amino acid residue isrequired for PKSRP activity. Other amino acid residues, however, (e.g.,those that are not conserved or only semi-conserved in the domain havingPKSRP activity) may not be essential for activity and thus are likely tobe amenable to alteration without altering PKSRP activity.

[0048] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding PKSRPs that contain changes in amino acidresidues that are not essential for PKSRP activity. Such PKSRPs differin amino acid sequence from a sequence contained in SEQ ID NO: 7, SEQ IDNO: 8 or SEQ ID NO: 9, yet retain at least one of the PKSRP activitiesdescribed herein. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 50% homologous to anamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and iscapable of participating in the a stress tolerance response in a plant,or more particularly participates in protein kinase signal transductionmechanisms involved in a stress tolerance response in a Physcomitrellapatens plant, or has one or more activities set forth in Table 1.Preferably, the protein encoded by the nucleic acid molecule is at leastabout 50-60% homologous to one of the sequences of SEQ ID NO: 7, SEQ IDNO: 8 and SEQ ID NO: 9, more preferably at least about 60-70% homologousto one of the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9,even more preferably at least about 70-80%, 80-90%, 90-95% homologous toone of the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, andmost preferably at least about 96%, 97%, 98%, or 99% homologous to oneof the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0049] To determine the percent homology of two amino acid sequences(e.g., one of the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:9and a mutant form thereof) or of two nucleic acids, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inthe sequence of one protein or nucleic acid for optimal alignment withthe other protein or nucleic acid). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in one sequence (e.g., oneof the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9) isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the other sequence (e.g., a mutant form of thesequence selected from the polypeptide of SEQ ID NO: 7, SEQ ID NO: 8 orSEQ ID NO: 9), then the molecules are homologous at that position (i.e.,as used herein amino acid or nucleic acid “homology” is equivalent toamino acid or nucleic acid “identity”). The percent homology between thetwo sequences is a function of the number of identical positions sharedby the sequences (i.e., % homology=numbers of identical positions/totalnumbers of positions×100). Preferably, the length of sequence comparisonis at least 15 amino acid residues, more preferably at least 25 aminoacid residues, and most preferably at least 35 amino acid residues.

[0050] Alternatively, a determination of the percent homology betweentwo sequences can be accomplished using a mathematical algorithm. Apreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of two sequences is the algorithm of Karlin and Altschul(1990 Proc. Natl. Acad. Sci. USA 90:5873-5877). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990 J. Mol. Biol. 215:403-410). BLAST nucleic acid searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleic acid sequences homologous to PKSRP nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to PKSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used to obtain amino acid sequenceshomologous to the PKSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used.

[0051] An isolated nucleic acid molecule encoding a PKSRP homologous toa protein sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5 orSEQ ID NO: 6 such that one or more amino acid substitutions, additionsor deletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of SEQ ID NO: 4, SEQ ID NO: 5 andSEQ ID NO: 6 by standard techniques, such as site-directed mutagenesisand PCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a PKSRPis preferably replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a PKSRP coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for a PKSRP activity described herein to identify mutants thatretain PKSRP activity. Following mutagenesis of one of the sequences ofSEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined by analyzing the stress tolerance of a plant expressing theprotein as described in Example 7.

[0052] In addition to the nucleic acid molecules encoding PKSRPsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules that arc antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence that is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire PKSRP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a PKSRP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the entire coding region of . . . comprises nucleotides 1 to . . . ). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding PKSRP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0053] Given the coding strand sequences encoding PKSRP disclosed herein(e.g., the sequences set forth in SEQ I) NO: 4, SEQ ID NO: 5 and SEQ IDNO: 6), antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof PKSRP mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of PKSRPmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of PKSRP mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0054] The antisense nucleic acid molecules of the invention aretypically administered to a cell or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aPKSRP to thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoters are preferred.

[0055] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al., 1987 Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987 Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987 FEBSLett. 215:327-330).

[0056] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff and Gerlach, 1988 Nature 334:585-591)) can be used tocatalytically cleave PKSRP mRNA transcripts to thereby inhibittranslation of PKSRP mRNA. A ribozyme having specificity for aPKSRP-encoding nucleic acid can be designed based upon the nucleotidesequence of a PKSRP cDNA disclosed herein (i.e., SEQ ID NO: 4, SEQ IDNO: 5 or SEQ ID NO: 6) or on the basis of a heterologous sequence to beisolated according to methods taught in this invention. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an PKSRP-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, PKSRP mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W., 1993 Science261:1411-1418.

[0057] Alternatively, PKSRP gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofa PKSRP nucleotide sequence (e.g., a PKSRP promoter and/or enhancer) toform triple helical structures that prevent transcription of an PKSRPgene in target cells. See generally, Helene, C., 1991 Anticancer DrugDes. 6(6):569-84; Helene, C. et al., 1992 Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J., 1992 Bioassays 14(12):807-15.

[0058] The invention further provides an isolated recombinant expressionvector comprising a nucleic acid as described above, wherein expressionof the vector in a host cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

[0059] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) or see: Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7,89-108, CRC Press: Boca Raton, Fla., including the references therein.Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence in many types of host cells and those thatdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded by nucleicacids as described herein (e.g., PKSRPs, mutant forms of PKSRPs, fusionproteins, etc.).

[0060] The recombinant expression vectors of the invention can bedesigned for expression of PKSRPs in prokaryotic or eukaryotic cells.For example, PKSRP genes can be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells (see Romanos, M. A. et al., 1992 Foreign geneexpression in yeast: a review, Yeast 8:423-488; van den Hondel, C. A. M.J. J. et al., 1991 Heterologous gene expression in filamentous fungi,in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds.,p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J.J. & Punt, P. J., 1991 Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae(Falciatore et al., 1999 Marine Biotechnology 1(3):239-251), ciliates ofthe types: Holotrichia, Peritrichia, Spirotrichia, Suctoria,Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in WO 98/01572 and multicellular plant cells (seeSchmidt, R. and Willmitzer, L., 1988 High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants, Plant Cell Rep. 583-586); Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung und R.Wu, 128-43, Academic Press: 1993; Potrykus, 1991 Annu. Rev. PlantPhysiol. Plant Molec. Biol. 42:205-225 and references cited therein) ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press:San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

[0061] Expression of proteins in prokaryotes is most often carried outwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, usually to theamino terminus of the recombinant protein but also to the C-terminus orfused within suitable regions in the proteins. Such fusion vectorstypically serve three purposes: 1) to increase expression of recombinantprotein; 2) to increase the solubility of the recombinant protein; and3) to aid in the purification of the recombinant protein by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

[0062] Typical fusion expression vectors include pGEX (Pharmacia BiotechInc; Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the PKSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant PKSRPunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

[0063] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., 1988 Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a co-expressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0064] One strategy to maximize recombinant protein expression is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asC. glutamicum (Wada et al., 1992 Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

[0065] In another embodiment, the PKSRP expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., 1987 Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, 1982 Cell 30:933-943), pJRY88 (Schultz etal., 1987 Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Vectors and methods for the construction of vectorsappropriate for use in other fungi, such as the filamentous fungi,includes those detailed in: van den Hondel, C. A. M. J. J. & Punt, P. J.(1991) “Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al.,eds., p. 1-28, Cambridge University Press: Cambridge.

[0066] Alternatively, the PKSRPs of the invention can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable, for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983 Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology170:31-39).

[0067] In yet another embodiment, a nucleic acid of the invent ion isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987Nature 329:840) and pMT2PC (Kaufman et al., 1987 EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0068] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al., 1987 Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton, 1988 Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore, 1989 EMBO J. 8:729-733) and immunoglobulins (Banerji et al.,1983 Cell 33:729-740; Queen and Baltimore, 1983 Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle, 1989 PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal., 1985 Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example the murine hox promoters(Kessel and Gruss, 1990 Science 249:374-379) and the fetoproteinpromoter (Campes and Tilghman, 1989 Genes Dev. 3:537-546).

[0069] In another embodiment, the PKSRPs of the invention may beexpressed in unicellular plant cells (such as algae) (see Falciatore etal., 1999 Marine Biotechnology 1(3):239-251 and references therein) andplant cells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992 New plantbinary vectors with selectable markers located proximal to the leftborder, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nuel. Acid. Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press, 1993, S. 15-38.

[0070] A plant expression cassette preferably contains regulatorysequences capable of driving gene expression in plants cells and whichare operably linked so that each sequence can fulfill its function, forexample, termination of transcription by polyadenylation signals.Preferred polyadenylation signals are those originating fromAgrobacterium tumefaciens t-DNA such as the gene 3 known as octopinesynthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984 EMBO J. 3:835)or functional equivalents thereof but also all other terminatorsfunctionally active in plants are suitable.

[0071] As plant gene expression is very often not limited ontranscriptional levels, a plant expression cassette preferably containsother operably linked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

[0072] Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and WO8402913) or plant promoters likethose from Rubisco small subunit described in U.S. Pat. No. 4962028.

[0073] Other preferred sequences for use in plant gene expressioncassettes are targeting-sequences necessary to direct the gene productin its appropriate cell compartment (for review see Kermode, 1996 Crit.Rev. Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells. Plant gene expression can also be facilitated via aninducible promoter (for review see Gatz, 1997 Annu. Rev. Plant Physiol.Plant Mol. Biol. 48:89-108). Chemically inducible promoters areespecially suitable if gene expression is wanted to occur in a timespecific manner. Examples of such promoters are a salicylic acidinducible promoter (WO 95/19443), a tetracycline inducible promoter(Gatz et al., 1992 Plant J. 2:397-404) and an ethanol inducible promoter(WO 93/21334).

[0074] Also, suitable promoters responding to biotic or abiotic stressconditions are those such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993 Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII-promoter (EP 375091). For other examples of drought, cold, andsalt-inducible promoters, such as the RD29A promoter, seeYamaguchi-Shinozalei et al. (1993 Mol. Gen. Genet. 236:331-340).

[0075] Especially those promoters are preferred which confer geneexpression in specific tissues and organs, such as guard cells and theroot hair cells. Suitable promoters include the napin-gene promoter fromrapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba(Baeumlein et al., 1991 Mol Gen Genet. 225(3):459-67), theoleosin-promoter from Arabidopsis (WO9845461), the phaseolin-promoterfrom Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoterfrom Brassica (WO9113980) or the legumin B4 promoter (LeB4; Bacumlein etal., 1992 Plant Journal, 2(2):233-9) as well as promoters conferringseed specific expression in monocot plants like maize, barley, wheat,rye, rice, etc. Suitable promoters to note are the lpt2 or lpt1-genepromoter from barley (WO 95/15389 and WO 95/23230) or those desribed inWO 99/16890 (promoters from the barley hordein-gene, rice glutelin gene,rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelingene, maize zein gene, oat glutelin gene, Sorghum kasirin-gene and ryesecalin gene).

[0076] Also especially suited are promoters that confer plastid-specificgene expression as plastids are the compartment where precursors andsome end products of lipid biosynthesis are synthesized. Suitablepromoters are the viral RNA-polymerase promoter described in WO 95/16783and WO 97/06250 and the c1pP-promoter from Arabidopsis described in WO99/46394.

[0077] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to PKSRP mRNA. Regulatory sequences operatively linkedto a nucleic acid molecule cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types. For instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews-Trends in Genetics, Vol. 1(1) 1986 and Mol et al., 1990 FEBSLetters 268:427-430.

[0078] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but they also apply to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0079] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a PKSRP can be expressed in bacterial cells such as C.glutamicum, insect cells, fungal cells or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plantcells, fungi or other microorganisms like C. glutamicum. Other suitablehost cells are known to those skilled in the art.

[0080] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation”, “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer and electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) and other laboratory manuals such asMethods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols,ed: Gartland and Davey, Humana Press, Totowa, N.J. As biotic and abioticstress tolerance is a general trait wished to be inherited into a widevariety of plants like maize, wheat, rye, oat, triticale, rice, barley,soybean, peanut, cotton, rapeseed and canola, manihot, pepper, sunflowerand tagetes, solanaceous plants like potato, tobacco, eggplant, andtomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea),Salix species, trees (oil palm, coconut), perennial grasses and foragecrops, these crops plants are also preferred target plants for a geneticengineering as one further embodiment of the present invention.

[0081] In particular, the invention provides a method of producing atransgenic plant with a PKSRP coding nucleic acid, wherein expression ofthe nucleic acid in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a PKSRP nucleic acid, and (b) generating from the plant cella transgenic plant with a increased tolerance to environmental stress ascompared to a wild type variety of the plant. In preferred embodiments,the PKSRP is as described above. In preferred embodiments, the PKSRPcoding nucleic acid is as described above. The invention also provides amethod of increasing expression of a gene of interest within a host cellas compared to a wild type variety of the host cell, wherein the gene ofinterest is transcribed in response to a PKSRP, comprising: (a)transforming the host cell with an expression vector comprising a PKSRPcoding nucleic acid, and (b) expressing the PKSRP within the host cell,thereby increasing the expression of the gene transcribed in response tothe PKSRP as compared to a wild type variety of the host cell. Inpreferred embodiments, the PKSRP is as described above. In preferredembodiments, the PKSRP coding nucleic acid is as described above.

[0082] For such plant transformation, binary vectors such as pBinAR canbe used (Höfgen and Willmitzer, 1990 Plant Science 66:221-230).Construction of the binary vectors can be performed by ligation of thecDNA in sense or antisense orientation into the T-DNA. 5-prime to thecDNA a plant promoter activates transcription of the cDNA. Apolyadenylation sequence is located 3-prime to the cDNA. Tissue-specificexpression can be archived by using a tissue specific promoter. Forexample, seed-specific expression can be archived by cloning the napinor LeB4 or USP promoter 5-prime to the cDNA. Also, any other seedspecific promoter element can be used. For constitutive expressionwithin the whole plant, the CaMV 35S promoter can be used. The expressedprotein can be targeted to a cellular compartment using a signalpeptide, for example for plastids, mitochondria or endoplasmic reticulum(Kermode, Crit. Rev. Plant Sci., 1996 4 (15):285-423). The signalpeptide is cloned 5-prime in frame to the cDNA to archive subcellularlocalization of the fusion protein. Agrobacterium mediated planttransformation can be performed using for example the GV3101(pMP90)(Koncz and Schell, 1986 Mol. Gen. Genet. 204:383-396) or LBA4404(Clontech) Agrobacterium tumefaciens strain. Transformation can beperformed by standard transformation techniques (Deblaere et al., 1994Nucl. Acids. Res. 13:4777-4788). In one embodiment, promoters that areresponsive to abiotic stresses can be used with, such as the Arabidopsispromoter RD29A, the nucleic acid sequences disclosed herein. One skilledin the art will recognize that the promoter used should be operativelylinked to the nucleic acid such that the promoter causes transcriptionof the nucleic acid which results in the synthesis of a mRNA whichencodes a polypeptide. Alternatively, the RNA can be an antisense RNAfor use in affecting subsequent expression of the same or another geneor genes.

[0083] Agrobacterium mediated plant transformation can be performedusing standard transformation and regeneration techniques (Gelvin,Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual,2nd Ed.—Dordrecht: Kluwer Academic Publ., 1995.—in Sect., RingbucZentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R.;Thompson, John E., Methods in Plant Molecular Biology and Biotechnology,Boca Raton : CRC Press, 1993.—360 S.,ISBN 0-8493-5164-2). For example,rapeseed can be transformed via cotyledon or hypocotyl transformation(Moloney et al., 1989 Plant cell Report 8:238-242; De Block et al., 1989Plant Physiol. 91:694-701). Use of antibiotica for Agrobacterium andplant selection depends on the binary vector and the Agrobacteriumstrain used for transformation. Rapeseed selection is normally performedusing kanamycin as selectable plant marker. Agrobacterium mediated genetransfer to flax can be performed using, for example, a techniquedescribed by Mlynarova et al., 1994 Plant Cell Report 13: 282-285.Additionally, transformation of soybean can be performed using forexample a technique described in EP 0424 047, U.S. Pat. No. 5,322,783(Pioneer Hi-Bred International) or in EP 0397 687, U.S. Pat. No.5,376,543, U.S. Pat. No. 5,169,770 (University Toledo).

[0084] Plant transformation using particle bombardment, PolyethyleneGlycol mediated DNA uptake or via the Silicon Carbide Fiber technique isfor example described by Freeling and Walbot “The maize handbook”Springer Verlag: New York (1993) ISBN 3-540-97826-7. A specific exampleof maize transformation is found in U.S. Pat. No. 5,990,387 and aspecific example of wheat transformation can be found in WO 93/07256.

[0085] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate or inplants that confer resistance towards a herbicide such as glyphosate orglufosinate. Nucleic acid molecules encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding an PKSRPor can be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid molecule can be identified by, for example,drug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

[0086] To create a homologous recombinant microorganism, a vector isprepared which contains at least a portion of a PKSRP gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the PKSRP gene. Preferably, this PKSRP geneis a Physcomitrella patens PKSRP gene, but it can be a homologue from arelated plant or even from a mammalian, yeast, or insect source. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous PKSRP gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as aknock-out vector). Alternatively, the vector can be designed such that,upon homologous recombination, the endogenous PKSRP gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous PKSRP). To create a point mutation viahomologous recombination, DNA-RNA hybrids can be used in a techniqueknown as chimeraplasty (Cole-Strauss et al., 1999 Nucleic Acids Research27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist.87(3):240-247). Homologous recombination procedures in Physcomitrellapatens are also well known in the art and are contemplated for useherein.

[0087] Whereas in the homologous recombination vector, the alteredportion of the PKSRP gene is flanked at its 5′ and 3′ ends by additionalnucleic acid molecule of the PKSRP gene to allow for homologousrecombination to occur between the exogenous PKSRP gene carried by thevector and an endogenous PKSRP gene in a microorganism or plant. Theadditional flanking PKSRP nucleic acid molecule is of sufficient lengthfor successful homologous recombination with the endogenous gene.Typically, several hundreds of base pairs up to kilobases of flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see e.g.,Thomas, K. R., and Capecchi, M. R., 1987 Cell 51:503 for a descriptionof homologous recombination vectors or Strepp et al., 1998 PNAS, 95(8):4368-4373 for cDNA based recombination in Physcomitrella patens).The vector is introduced into a microorganism or plant cell (e.g., viapolyethylene glycol mediated DNA) and cells in which the introducedPKSRP gene has homologously recombined with the endogenous PKSRP geneare selected, using art-known techniques.

[0088] In another embodiment, recombinant microorganisms can be producedwhich contain selected systems which allow for regulated expression ofthe introduced gene. For example, inclusion of a PKSRP gene on a vectorplacing it under control of the lac operon permits expression of thePKSRP gene only in the presence of IPTG. Such regulatory systems arewell known in the art.

[0089] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a PKSRP. Analternate method can be applied in addition in plants by the directtransfer of DNA into developing flowers via electroporation orAgrobacterium medium gene transfer. Accordingly, the invention furtherprovides methods for producing PKSRPs using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of invention (into which a recombinant expression vector encoding aPKSRP has been introduced, or into which genome has been introduced agene encoding a wild-type or altered PKSRP) in a suitable medium untilPKSRP is produced. In another embodiment, the method further comprisesisolating PKSRPs from the medium or the host cell.

[0090] Another aspect of the invention pertains to isolated PKSRPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is free of some of thecellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof PKSRP in which the protein is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of PKSRP having less than about30% (by dry weight) of non-PKSRP (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-PKSRP, still more preferably less than about 10% of non-PKSRP, andmost preferably less than about 5% non-PKSRP. When the PKSRP orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation. The language “substantially free of chemical precursors orother chemicals” includes preparations of PKSRP in which the protein isseparated from chemical precursors or other chemicals that are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of PKSRP having less than about 30% (by dry weight) ofchemical precursors or non-PKSRP chemicals, more preferably less thanabout 20% chemical precursors or non-PKSRP chemicals, still morepreferably less than about 10% chemical precursors or non-PKSRPchemicals, and most preferably less than about 5% chemical precursors ornon-PKSRP chemicals. In preferred embodiments, isolated proteins orbiologically active portions thereof lack contaminating proteins fromthe same organism from which the PKSRP is derived. Typically, suchproteins are produced by recombinant expression of, for example, aPhyscomitrella patens PKSRP in plants other than Physcomitrella patensor microorganisms such as C. glutamicum, ciliates, algae or fungi.

[0091] An isolated PKSRP or a portion thereof of the invention canparticipate in a stress tolerance response in a plant, or moreparticularly can participate in the protein kinase signal transductionmechanisms involved in a stress tolerance response in a Physcomitrellapatens plant, or has one or more of the activities set forth in Table 1.In preferred embodiments, the protein or portion thereof comprises anamino acid sequence which is sufficiently homologous to an amino acidsequence encoded by a nucleic acid of SEQ ID NO: 4, SEQ ID NO: 5 or SEQID NO: 6 such that the protein or portion thereof maintains the abilityto participate in the metabolism of compounds necessary for theconstruction of cellular membranes in Physcomitrella patens, or in thetransport of molecules across these membranes. The portion of theprotein is preferably a biologically active portion as described herein.In another preferred embodiment, a PKSRP of the invention has an aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. In yetanother preferred embodiment, the PKSRP has an amino acid sequence whichis encoded by a nucleotide sequence which hybridizes, e.g., hybridizesunder stringent conditions, to a nucleotide sequence of SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO: 6. In still another preferred embodiment, thePKSRP has an amino acid sequence which is at least about 50-60%,preferably at least about 60-70%, more preferably at least about 70-80%,80-90%, 90-95%, and even more preferably at least about 96%, 97%, 98%,99% or more homologous to one of the amino acid sequences of SEQ ID NO:7, SEQ ID NO: 8 and SEQ ID NO: 9. The preferred PKSRPs of the presentinvention also preferably possess at least one of the PKSRP activitiesdescribed herein. For example, a preferred PKSRP of the presentinvention includes an amino acid sequence encoded by a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6,and which can participate can participate in a stress tolerance responsein a plant, or more particularly can participate in the transcription ofa protein involved in a stress tolerance response in a Physcomitrellapatens plant, or which has one or more of the activities set forth inTable 1.

[0092] In other embodiments, the PKSRP is substantially homologous to anamino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 andretains the functional activity of the protein of one of the sequencesof SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, yet differs in aminoacid sequence due to natural variation or mutagenesis, as described indetail above. Accordingly, in another embodiment, the PKSRP is a proteinwhich comprises an amino acid sequence which is at least about 50-60%,preferably at least about 60-70%, and more preferably at least about70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%,99% or more homologous to an entire amino acid sequence of SEQ ID NO: 7,SEQ ID NO: 8 or SEQ ID NO: 9 and which has at least one of the PKSRPactivities described herein. In another embodiment, the inventionpertains to a full Physcomitrella patens protein which is substantiallyhomologous to an entire amino acid sequence encoded by a nucleic acid ofSEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0093] Biologically active portions of an PKSRP include peptidescomprising amino acid sequences derived from the amino acid sequence ofan PKSRP, e.g., an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 orSEQ ID NO: 9 or the amino acid sequence of a protein homologous to anPKSRP, which include fewer amino acids than a full length PKSRP or thefull length protein which is homologous to an PKSRP, and exhibit atleast one activity of an PKSRP. Typically, biologically active portions(peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35,36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise adomain or motif with at least one activity of a PKSRP. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the activities described herein. Preferably, the biologicallyactive portions of a PKSRP include one or more selected domains/motifsor portions thereof having biological activity.

[0094] PKSRPs are preferably produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the PKSRP isexpressed in the host cell. The PKSRP can then be isolated from thecells by an appropriate purification scheme using standard proteinpurification techniques. Alternative to recombinant expression, a PKSRP,polypeptide, or peptide can be synthesized chemically using standardpeptide synthesis techniques. Moreover, native PKSRP can be isolatedfrom cells (e.g., Physcomitrella patens), for example using ananti-PKSRP antibody, which can be produced by standard techniquesutilizing a PKSRP or fragment thereof of this invention.

[0095] The invention also provides PKSRP chimeric or fusion proteins. Asused herein, a PKSRP “chimeric protein” or “fusion protein” comprises aPKSRP polypeptide operatively linked to a non-PKSRP polypeptide. An“PKSRP polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a PKSRP, whereas a “non-PKSRP polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the PKSRP, e.g., aprotein which is different from the PKSRP and which is derived from thesame or a different organism. Within the fusion protein, the term“operatively linked” is intended to indicate that the PKSRP polypeptideand the non-PKSRP polypeptide are fused to each other so that bothsequences fulfill the proposed function attributed to the sequence used.The non-PKSRP polypeptide can be fused to the N-terminus or C-terminusof the PKSRP polypeptide. For example, in one embodiment, the fusionprotein is a GST-PKSRP fusion protein in which the PKSRP sequences arefused to the C-terminus of the GST sequences. Such fusion proteins canfacilitate the purification of recombinant PKSRPs. In anotherembodiment, the fusion protein is a PKSRP containing a heterologoussignal sequence at its N-terminus. In certain host cells (e.g.,mammalian host cells), expression and/or secretion of a PKSRP can beincreased through use of a heterologous signal sequence.

[0096] Preferably, a PKSRP chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). APKSRP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the PKSRP.

[0097] Homologues of the PKSRP can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the PKSRP. As used herein, theterm “homologue” refers to a variant form of the PKSRP which acts as anagonist or antagonist of the activity of the PKSRP. An agonist of thePKSRP can retain substantially the same, or a subset, of the biologicalactivities of the PKSRP. An antagonist of the PKSRP can inhibit one ormore of the activities of the naturally occurring form of the PKSRP, by,for example, competitively binding to a downstream or upstream member ofthe cell membrane component metabolic cascade which includes the PKSRP,or by binding to an PKSRP which mediates transport of compounds acrosssuch membranes, thereby preventing translocation from taking place.

[0098] In an alternative embodiment, homologues of the PKSRP can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the PKSRP for PKSRP agonist or antagonistactivity. In one embodiment, a variegated library of PKSRP variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of PKSRPvariants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential PKSRP sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of PKSRP sequences therein.There are a variety of methods which can be used to produce libraries ofpotential PKSRP homologues from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene is then ligated intoan appropriate expression vector. Use of a degenerate set of genesallows for the provision, in one mixture, of all of the sequencesencoding the desired set of potential PKSRP sequences. Methods forsynthesizing degenerate oligonucleotides are known in the art (see,e.g., Narang, S. A., 1983 Tetrahedron 39:3; Itakura et al., 1984 Annu.Rev. Biochem. 53:323; Itakura et al., 1984 Science 198:1056; Ike et al.,1983 Nucleic Acid Res. 11:477.

[0099] In addition, libraries of fragments of the PKSRP coding can beused to generate a variegated population of PKSRP fragments forscreening and subsequent selection of homologues of a PKSRP. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a PKSRP coding sequence witha nuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PKSRP.

[0100] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of PKSRPhomologues. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify PKSRP homologues (Arkin and Yourvan, 1992PNAS 89:7811-7815; Delgrave et al., 1993 Protein Engineering6(3):327-331). In another embodiment, cell based assays can be exploitedto analyze a variegated PKSRP library, using methods well known in theart. The present invention further provides a method of identifying anovel PKSRP, comprising (a) raising a specific antibody response to aPKSRP, or fragment thereof, as described above; (b) screening putativePKSRP material with the antibody, wherein specific binding of theantibody to the material indicates the presence of a potentially novelPKSRP; and (c) analyzing the bound material in comparison to known PKSRPto determine its novelty.

[0101] The nucleic acid molecules, proteins, protein homologues, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification ofPhyscomitrella patens and related organisms; mapping of genomes oforganisms related to Physcomitrella patens; identification andlocalization of Physcomitrella patens sequences of interest;evolutionary studies; determination of PKSRP regions required forfunction; modulation of an PKSRP activity; modulation of the metabolismof one or more cell functions; modulation of the transmembrane transportof one or more compounds; and modulation of stress resistance.

[0102] The moss Physcomitrella patens represents one member of themosses. It is related to other mosses such as Ceratodon purpureus whichis capable of growth in the absence of light. Mosses like Ceratodon andPhyscomitrella share a high degree of homology on the DNA sequence andpolypeptide level allowing the use of heterologous screening of DNAmolecules with probes evolving from other mosses or organisms, thusenabling the derivation of a consensus sequence suitable forheterologous screening or functional annotation and prediction of genefunctions in third species. The ability to identify such functions cantherefore have significant relevance, e.g., prediction of substratespecificity of enzymes. Further, these nucleic acid molecules may serveas reference points for the mapping of moss genomes, or of genomes ofrelated organisms.

[0103] The PKSRP nucleic acid molecules of the invention have a varietyof uses. Most importantly, the nucleic acid and amino acid sequences ofthe present invention can be used to transform plants, thereby inducingtolerance to stresses such as drought, high salinity and cold. Thepresent invention therefore provides a transgenic plant transformed by aPKSRP coding nucleic acid, wherein expression of the nucleic acidsequence in the plant results in increased tolerance to environmentalstress as compared to a wild type variety of the plant. The transgenicplant can be a monocot or a dicot. The invention further provides thatthe transgenic plant can be selected from maize, wheat, rye, oat,triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola,manihot, pepper, sunflower, tagetes, solanaceous plants, potato,tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,tea, Salix species, oil palm, coconut, perennial grass and forage crops,for example. In particular, the present invention describes using theexpression of PK-1 (SEQ ID NO: 7), PK-2 (SEQ ID NO: 8) and MPK-1 (SEQ IDNO: 9) to engineer drought-tolerant plants. This strategy has hereinbeen demonstrated for Arabidopsis thaliana, Rapeseed/Canola, soybeans,corn and wheat but its application is not restricted to these plants.Accordingly, the invention provides a transgenic plant containing aPKSRP selected from 1) PK-1; 2) PK-2; 3) PK-3 as defined above,including homologues, wherein the environmental stress is drought. Thisinvention also describes the principle of using over-expression of PK-2(SEQ ID NO: 8) and MPK-1 (SEQ ID NO: 9) to engineer salt-tolerantplants. Again, this strategy has herein been demonstrated forArabidopsis thaliana, Rapeseed/Canola, soybeans, corn and wheat but itsapplication is not restricted to these plants. Accordingly, theinvention provides a transgenic plant containing the PKSRP selectedfrom 1) PK-2 and 2) PK-3 as defined above, including homologues, whereinthe environmental stress is salinity.

[0104] The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a PKSRP inthe plant. The invention provides that this method can be performed suchthat the stress tolerance is either increased or decreased.

[0105] Furthermore, this method can be used wherein the plant is eithertransgenic or not transgenic. In cases when the plant is transgenic, theplant can be transformed with a vector containing any of the abovedescribed PKSRP coding nucleic acids, or the plant can be transformedwith a promoter that directs expression of native PKSRP in the plant,for example. The invention provides that such a promoter can be tissuespecific. Furthermore, such a promoter can be developmentally regulated.Alternatively, non-transgenic plants can have native PKSRP expressionmodified by inducing a native promoter. Furthermore, the inventionprovides that PKSRP expression can be modified by administration of ananti-sense molecule that inhibits expression of PKSRP.

[0106] The expression of PK-1 (SEQ ID NO: 7), PK-2 (SEQ ID NO: 8) andMPK-1 (SEQ ID NO: 9) in target plants can be accomplished by, but is notlimited to, one of the following examples: (a) constitutive promoter,(b) stress-inducible promoter, (c) chemical-induced promoter, and (d)engineered promoter over-expression with for example zinc-finger derivedtranscription factors (Greisman and Pabo, 1997 Science 275:657). Thelater case involves identification of the PK-1 (SEQ ID NO: 7), PK-2 (SEQID NO: 8) and MPK-1 (SEQ ID NO: 9) homologues in the target plant aswell as from its promoter. Zinc-finger-containing recombinanttranscription factors are engineered to specifically interact with thePK-1 (SEQ ID NO: 7), PK-2 (SEQ ID NO: 8) and MPK-1 (SEQ ID NO: 9)homologue and transcription of the corresponding gene is activated.

[0107] As shown herein and described more fully below, expression of thePKSRPs (PK-1 (SEQ ID NO: 7), PK-2 (SEQ ID NO: 8) and MPK-1 (SEQ ID NO:9)) in Arabidopsis thaliana confers a high degree of drought toleranceto the plant. Additionally, several PKSRPs confer tolerance to high saltconcentrations (PK-2 (SEQ ID NO: 8) and MPK-1 (SEQ ID NO: 9)) to thisplant.

[0108] In addition to introducing the PKSRP nucleic acid sequences intotransgenic plants, these sequences can also be used to identify anorganism as being Physcomitrella patens or a close relative thereof.Also, they may be used to identify the presence of Physcomitrella patensor a relative thereof in a mixed population of microorganisms. Theinvention provides the nucleic acid sequences of a number ofPhyscomitrella patens genes; by probing the extracted genomic DNA of aculture of a unique or mixed population of microorganisms understringent conditions with a probe spanning a region of a Physcomitrellapatens gene which is unique to this organism, one can ascertain whetherthis organism is present.

[0109] Further, the nucleic acid and protein molecules of the inventionmay serve as markers for specific regions of the genome. This hasutility not only in the mapping of the genome, but also in functionalstudies of Physcomitrella patens proteins. For example, to identify theregion of the genome to which a particular Physcomitrella patensDNA-binding protein binds, the Physcomitrella patens genome could bedigested, and the fragments incubated with the DNA-binding protein.Those which bind the protein may be additionally probed with the nucleicacid molecules of the invention, preferably with readily detectablelabels; binding of such a nucleic acid molecule to the genome fragmentenables the localization of the fragment to the genome map ofPhyscomitrella patens, and, when performed multiple times with differentenzymes, facilitates a rapid determination of the nucleic acid sequenceto which the protein binds. Further, the nucleic acid molecules of theinvention may be sufficiently homologous to the sequences of relatedspecies such that these nucleic acid molecules may serve as markers forthe construction of a genomic map in related mosses.

[0110] The PKSRP nucleic acid molecules of the invention are also usefulfor evolutionary and protein structural studies. The metabolic andtransport processes in which the molecules of the invention participateare utilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein which are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

[0111] Manipulation of the PKSRP nucleic acid molecules of the inventionmay result in the production of PKSRPs having functional differencesfrom the wild-type PKSRPs. These proteins may be improved in efficiencyor activity, may be present in greater numbers in the cell than isusual, or may be decreased in efficiency or activity. There are a numberof mechanisms by which the alteration of a PKSRP of the invention maydirectly affect stress response and/or stress tolerance. In the case ofplants expressing PKSRPs, increased transport can lead to improved saltand/or solute partitioning within the plant tissue and organs. By eitherincreasing the number or the activity of transporter molecules whichexport ionic molecules from the cell, it may be possible to affect thesalt tolerance of the cell.

[0112] The effect of the genetic modification in plants, C. glutamicum,fungi, algae, or ciliates on stress tolerance can be assessed by growingthe modified microorganism or plant under less than suitable conditionsand then analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, protein synthesis, carbohydratesynthesis, lipid synthesis, evapotranspiration rates, general plantand/or crop yield, flowering, reproduction, seed setting, root growth,respiration rates, photosynthesis rates, etc. (Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III:Product recovery and purification, page 469-714, VCH: Weinheim; Belter,P. A. et al., 1988 Bioseparations: downstream processing forbiotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S.,1992 Recovery processes for biological materials, John Wiley and Sons;Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemical separations, in:Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications).

[0113] For example, yeast expression vectors comprising the nucleicacids disclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived therefrom can then be assayed forfail or alteration of their tolerance to drought, salt, and temperaturestress.

[0114] The engineering of one or more PKSRP genes of the invention mayalso result in PKSRPs having altered activities which indirectly impactthe stress response and/or stress tolerance of algae, plants, ciliatesor fungi or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more PKSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

[0115] Additionally, the sequences disclosed herein, or fragmentsthereof, can be used to generate knockout mutations in the genomes ofvarious organisms, such as bacteria, mammalian cells, yeast cells, andplant cells. (Girke, T., 1998 The Plant Journal 15:39-48). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation include U.S. Pat. No.6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999Spliceosome-mediated RNA trans-splicing as a tool for gene therapyNature Biotechnology 17:246-252.

[0116] The aforementioned mutagenesis strategies for PKSRPs to result inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and protein molecules of the invention may beutilized to generate algae, ciliates, plants, fungi or othermicroorganisms like C. glutamicum expressing mutated PKSRP nucleic acidand protein molecules such that the stress tolerance is improved.

[0117] The present invention also provides antibodies which specificallybind to a PKSRP-polypeptide, or a portion thereof, as encoded by anucleic acid disclosed herein or as described herein. Antibodies can bemade by many well-known methods (See, e.g. Harlow and Lane, “Antibodies;A Laboratory Manual” Cold Spring Harbor Laboratory, Cold Spring Harbor,New York, (1988)). Briefly, purified antigen can be injected into ananimal in an amount and in intervals sufficient to elicit an immuneresponse. Antibodies can either be purified directly, or spleen cellscan be obtained from the animal. The cells can then fused with animmortal cell line and screened for antibody secretion. The antibodiescan be used to screen nucleic acid clone libraries for cells secretingthe antigen. Those positive clones can then be sequenced. (See, forexample, Kelly et al., 1992 Bio/Technology 10:163-167; Bebbington etal., 1992 Bio/Technology 10:169-175).

[0118] The phrases “selectively binds” and “specifically binds” with thepolypeptide refers to a binding reaction which is determinative of thepresence of the protein in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bound to a particular protein do not bind in asignificant amount to other proteins present in the sample. Selectivebinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats may be used to select antibodies selectively bindwith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select antibodies selectively immunoreactive witha protein. See Harlow and Lane “Antibodies, A Laboratory Manual” ColdSpring Harbor Publications, New York, (1988), for a description ofimmunoassay formats and conditions that could be used to determineselective binding.

[0119] In some instances, it is desirable to prepare monoclonalantibodies from various hosts. A description of techniques for preparingsuch monoclonal antibodies may be found in Stites et al., editors,“Basic and Clinical Immunology,” (Lange Medical Publications, Los Altos,Calif., Fourth Edition) and references cited therein, and in Harlow andLane (“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications,New York, 1988).

[0120] Throughout this application various publications are referenced.The disclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

[0121] It should also be understood that the foregoing relates topreferred embodiments of the present invention and that numerous changesmay be made therein without departing from the scope of the invention.The invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims. Additionally, all references cited herein are hereby expresslyincorporated herein by reference.

EXAMPLES Example 1

[0122] Growth of Physcomitrella patens Cultures

[0123] For this study, plants of the species Physcomitrella patens(Hedw.) B. S. G. from the collection of the genetic studies section ofthe University of Hamburg were used. They originate from the strain16/14 collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire(England), which was subcultured from a spore by Engel (1968, Am. J.Bot. 55, 438-446). Proliferation of the plants was carried out by meansof spores and by means of regeneration of the gametophytes. Theprotonema developed from the haploid spore as a chloroplast-richchloronema and chloroplast-low caulonema, on which buds formed afterapproximately 12 days. These grew to give gametophores bearingantheridia and archegonia. After fertilization, the diploid sporophytewith a short seta and the spore capsule resulted, in which themeiospores matured.

[0124] Culturing was carried out in a climatic chamber at an airtemperature of 25° C. and light intensity of 55 micromols^(−1m2) (whitelight; Philips TL 65W/25 fluorescent tube) and a light/dark change of16/8 hours. The moss was either modified in liquid culture using Knopmedium according to Reski and Abel (1985, Planta 165:354-358) orcultured on Knop solid medium using 1% oxoid agar (Unipath, Basingstoke,England). The protonemas used for RNA and DNA isolation were cultured inaerated liquid cultures. The protonemas were comminuted every 9 days andtransferred to fresh culture medium.

Example 2

[0125] Total DNA Isolation From Plants

[0126] The details for the isolation of total DNA relate to the workingup of one gram fresh weight of plant material. The materials usedinclude the following buffers: CTAB buffer: 2% (w/v)N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0;1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v)N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

[0127] The plant material was triturated under liquid nitrogen in amortar to give a fine powder and transferred to 2 ml Eppendorf vessels.The frozen plant material was then covered with a layer of I ml ofdecomposition buffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosinebuffer, 20 μl of β-mercaptoethanol and 10 μl of proteinase K solution,10 mg/ml) and incubated at 60° C. for one hour with continuous shaking.The homogenate obtained was distributed into two Eppendorf vessels (2ml) and extracted twice by shaking with the same volume ofchloroform/isoamyl alcohol (24:1). For phase separation, centrifugationwas carried out at 8000×g and room temperature for 15 minutes in eachcase. The DNA was then precipitated at −70° C. for 30 min using ice-coldisopropanol. The precipitated DNA was sedimented at 4° C. and 10,000 gfor 30 minutes and resuspended in 180 μl of TE buffer (Sambrook et al.,1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). Forfurther purification, the DNA was treated with NaCl (1.2 M finalconcentration) and precipitated again at −70° C. for 30 minutes usingtwice the volume of absolute ethanol. After a washing step with 70%ethanol, the DNA was dried and subsequently taken up in 50 μl ofH₂O+RNAse (50 mg/ml final concentration). The DNA was dissolvedovernight at 4° C. and the RNAse digestion was subsequently carried outat 37° C. for 1 hour. Storage of the DNA took place at 4° C.

Example 3

[0128] Isolation of Total RNA and poly-(A)+RNA and cDNA LibraryConstruction From Physcomitrella patens

[0129] For the investigation of transcripts, both total RNA andpoly-(A)+RNA were isolated. The total RNA was obtained from wild-type 9day old protonemata following the GTC-method (Reski et al. 1994, Mol.Gen. Genet., 244:352-359). The Poly(A)+RNA was isolated using DynaBeads^(R) (Dynal, Oslo, Norway) following the instructions of themanufacturers protocol. After determination of the concentration of theRNA or of the poly(A)+RNA, the RNA was precipitated by addition of 1/10volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and storedat −70° C.

[0130] For cDNA library construction, first strand synthesis wasachieved using Murine Leukemia Virus reverse transcriptase (Roche,Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis byincubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at12° C. (2 hours), 16° C. (1 hour) and 22° C. (1 hour). The reaction wasstopped by incubation at 65° C. (10 minutes) and subsequentlytransferred to ice. Double stranded DNA molecules were blunted byT4-DNA-polymerase (Roche, Mannheim) at 37° C. (30 minutes). Nucleotideswere removed by phenol/chloroform extraction and Sephadex G50 spincolumns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated tothe cDNA ends by T4-DNA-ligase (Roche, 12° C., overnight) andphosphorylated by incubation with polynucleotide kinase (Roche, 37° C.,30 minutes). This mixture was subjected to separation on a low meltingagarose gel. DNA molecules larger than 300 base pairs were eluted fromthe gel, phenol extracted, concentrated on Elutip-D-columns (Schleicherand Schuell, Dassel, Germany) and were ligated to vector arms and packedinto lambda ZAPII phages or lambda ZAP-Express phages using the GigapackGold Kit (Stratagene, Amsterdam, Netherlands) using material andfollowing the instructions of the manufacturer.

Example 4

[0131] Sequencing and Function Annotation of Physcomitrella patens ESTs

[0132] cDNA libraries as described in Example 3 were used for DNAsequencing according to standard methods, and in particular, by thechain termination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random Sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via in vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands.Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (see Sambrook et al. 1989 ColdSpring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNApreparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used: 5′-CAGGAAACAGCTATGACC-3′ SEQ ID NO:105′-CTAAAGGGAACAAAAGCTG-3′ SEQ ID NO:11 5′-TGTAAAACGACGGCCAGT-3′ SEQ IDNO:12

[0133] Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference the website at pedant.mips.biochem.mpg.de. The most importantalgorithms incorporated in EST-MAX are: FASTA: Very sensitive sequencedatabase searches with estimates of statistical significance; Pearson W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA.Methods Enzymol. 183:63-98; BLAST: Very sensitive sequence databasesearches with estimates of statistical significance. Altschul S. F.,Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignmentsearch tool. Journal of Molecular Biology 215:403-10; PREDATOR:High-accuracy secondary structure prediction from single and multiplesequences. Frishman, D. and Argos, P. (1997) 75% accuracy in proteinsecondary structure prediction. Proteins, 27:329-335; CLUSTALW: Multiplesequence alignment. Thompson, J. D., Higgins, D. G. and Gibson, T. J.(1994) CLUSTALW: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Research,22:4673-4680; TMAP: Transmembrane region prediction from multiplyaligned sequences. Persson, B. and Argos, P. (1994) Prediction oftransmembrane segments in proteins utilizing multiple sequencealignments. J. Mol. Biol. 237:182-192; ALOM2: Transmembrane regionprediction from single sequences. Klein, P., Kanehisa, M., and DeLisi,C. Prediction of protein function from sequence properties: Adiscriminate analysis of a database. Biochim. Biophys. Acta 787:221-226(1984). Version 2 by Dr. K. Nakai; PROSEARCH: Detection of PROSITEprotein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M.,Smith J. E. (1992) ProSearch: fast searching of protein sequences withregular expression patterns related to protein structure and function.Biotechniques 13, 919-921; BLIMPS: Similarity searches against adatabase of ungapped blocks. J. C. Wallace and Henikoff S., (1992);PATMAT: A searching and extraction program for sequence, pattern andblock queries and databases, CABIOS 8:249-254. Written by Bill Alford.

Example 5

[0134] Identification of Physcomitrella patens ORF Corresponding toPpPK-1, PpPK-2 and PpMPK-1

[0135] The Physcomitrella patens partial cDNAs (ESTs) shown in Table 1below were identified in the Physcomitrella patens EST sequencingprogram using the program EST-MAX through BLAST analysis. (Tables 2-4show some of the results). The Sequence Identification Numberscorresponding to these ESTs are as follows: PpPK-1 (SEQ ID NO: 1);PpPK-2 (SEQ ID NO: 2) and PpMPK-1 (SEQ ID NO: 3). These particularclones were chosen for farther analyses since they encoded for proteinkinases. TABLE 1 Functional ORF Category Function Sequence Code positionName Protein MAP kinase s_pp004047334r 1-560 PpMPK-1 Kinaseserine/threonine s_pp004012036r 1-467 PpPK-2 protein kinaseserine/threonine s_pp004005088r 1-324 PpK-1 kinase

[0136] TABLE 2 Degree of Amino Acid Identity and Similarity of PpPK-1and Other Homologous Proteins (GCG Gap program was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum62) Swiss- Prot #O24342 Q9ZVD9 Q9MAM1 Q9LDI3 Q9LKC9 Protein Serine/ Putative T25K16.13Serine/ CBL- name Threonine Serine/ Threonine interacting kinaseThreonine protein protein protein kinase kinase 3 kinase SOS2 SpecieSorghum Arabid- Arabid- Arabid- Arabid- bicolor opsis opsis opsis opsis(Sorghum) thaliana thaliana thaliana thaliana (Mouse- (Mouse- (Mouse-(Mouse- ear cress) ear cress) ear cress) ear cress) Iden- 66% 62% 64%55% 55% tity % Simi- 78% 76% 75% 69% 67% larity %

[0137] TABLE 3 Degree of Amino Acid Identity and Similarity of PpPK-2and Other Homologous Proteins (GCG Gap program was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum62) Swiss- Prot #Q9LZ05 Q9LZ96 Q9LJU5 P43293 O04245 Protein Protein Serine/ ReceptorProbable Putative name kinase- Threonine- protein Serine/ NAK-like likespecific kinase- Threonine- Ser/Thr protein like protein protein kinaseprotein kinase kinase NAK NAK Specie Arabid- Arabid- Arabid- Arabid-Arabid- opsis opsi opsis opsis opsis thaliana thaliana thaliana thalianathaliana (Mouse- (Mouse- (Mouse- (Mouse- (Mouse- ear cress) ear cress)ear cress) ear cress) ear cress) Iden- 32% 32% 32% 32% 28% tity % Simi-43% 41% 41% 41% 35% larity %

[0138] TABLE 4 Degree of Amino Acid Identity and Similarity of PpMPK-1and Other Homologous Proteins (GCG Gap program was used: gap penalty:10; gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot #Q06060 Q39024 Q40532 Q9M136 Q9M534 Protein name Mitogen- Mitogen-Mitogen- MAP Mitogen- activated activated activated kinase 4 activatedprotein protein protein protein kinase kinase kinase kinase homologhomolog 4 homolog D5 NTF4 specie Pisum Arabid- Nicotiana Arabid-Euphorbi sativum opsis tabacum opsis aesula (Garden thaliana (Commonthaliana (Leafy pea) (Mouse- tobacco) (Mouse- spurge) ear cress) earcress) Identity % 45% 45% 44% 45% 44% Similarity % 53% 52% 53% 52% 53%

Example 6

[0139] Cloning of the Full-Length Physcomitrella patens cDNA Encodingfor PpPK-1, PpPK-2 and PpMPK-1

[0140] To isolate the clones encoding for PpPK-1, PpPk-2 and PpMPK-1from Physcomitrella patens, cDNA libraries were created with SMART RACEcDNA Amplification kit (Clontech Laboratories) following manufacturer'sinstructions. Total RNA isolated as described in Example 3 was used asthe template. The oligos designed for RACE are shown in Table 2. Thecultures were treated prior to RNA isolation as follows: Salt Stress: 2,6, 12, 24, 48 h with 1-M NaCl-supplemented medium; Cold Stress: 4° C.for the same time points as for salt; Drought Stress: cultures wereincubated on dry filter paper for the same time points above. TABLE 5Scheme and primers used for cloning of full-length clones Final sitesIsolation Gene in product Method Primers Race Primer RT-PCR PpPK-1XmaI/SacI 5′ RACE RC077 (SEQ ID NO:13) RC096 (SEQ ID NO:14) and RT-PCR5′CCGGGCTCCTGACGAGAA 5′ATCCCGGGCGTTCAAGCA for CCAAGGA GGTGAATATGACAACFull-length clone RC097 (SEQ ID NO:15) 3′ATGAGCTCGGGCACTTCACAGCACGGGCATAT PpPK-2 Xmal/HpaI 5′ RACE RC051 (SEQ ID NO:15) RC156 (SEQID NO:16) and RT-PCR 5′GCCCCCTTCGCCGACTAC 5′ATCCCGGGCGCGCACAAT forATTATCA3′ TTCAGTTGGGAATCA Full-length clone RC157 (SEQ ID NO:17)5′GCGTTAACGCCCGCAAAG GTCAAAACAGGCGTGG PpMPK-1 XmaI/HpaI 5′ RACE RC074(SEQ ID NO:18) RC100 (SEQ ID NO:19) and RT-PCR 5′GCCGCTGTGTACATTCGG5′ATCCCGGGCGGTTTGGAC for CTCCCAAG ACGATGTTCCAGTCC Full-length cloneRC101 (SEQ ID NO:20) 5′GCGTTAACTAACCGCGTT TAAGTCCCTCAAC

[0141] 5′ RACE Protocol

[0142] The EST sequences PpPK-1 (SEQ ID NO: 1), PpPK-2 (SEQ ID NO: 2)and PpMPK-1 (SEQ ID NO: 3) identified from the database search asdescribed in Example 4 were used to design oligos for RACE (see Table5). The extended sequences for these genes were obtained by performingRapid Amplification of cDNA Ends polymerase chain reaction (RACE PCR)using the Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACEcDNA amplification kit (Clontech Laboratories) using a Biometra T3Thermocycler following the manufacturer's instructions. The sequencesobtained from the RACE reactions corresponded to full-length codingregions of PpPK-1, PpPK-2 and PpMPK-1 and were used to design oligos forfull-length cloning of the respective genes (see below full-lengthamplification).

[0143] Full-length Amplification

[0144] Full-length clones of PpPK-1 (SEQ ID NO: 4), PpPK-2 (SEQ ID NO:5) and PpMPK-1 (SEQ ID NO: 6) were obtained by performing polymerasechain reaction (PCR) with gene-specific primers (see Table 5) and theoriginal EST as the template. The conditions for the reaction werestandard conditions with PWO DNA polymerase (Roche). PCR was performedaccording to standard conditions and to manufacturer's protocols(Sambrook et al. 1989. Molecular Cloning, A Laboratory Manual. 2ndEdition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.,Biometra T3 Thermocycler). The parameters for the reaction were: fiveminutes at 94° C followed by five cycles of one minute at 94° C., oneminute at 50° C. and 1.5 minutes at 72° C. This was followed by twentyfive cycles of one minute at 94° C., one minute at 65° C. and 1.5minutes at 72° C.

[0145] The amplified fragments were extracted from agarose gel with aQIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1vector (Invitrogen) following manufacturer's instructions. Recombinantvectors were transformed into Top10 cells (Invitrogen) using standardconditions (Sambrook et al. 1989. Molecular Cloning, A LaboratoryManual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold SpringHarbor, N.Y.). Transformed cells were selected for on LB agar containing100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside) grown overnight at 37° C. White colonieswere selected and used to inoculate 3 ml of liquid LB containing 100μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extractedusing the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer'sinstructions. Analyses of subsequent clones and restriction mapping wasperformed according to standard molecular biology techniques (Sambrooket al. 1989. Molecular Cloning, A Laboratory Manual. 2nd Edition. ColdSpring Harbor Laboratory Press. Cold Spring Harbor, N.Y.).

Example 7

[0146] Engineering Stress-Tolerant Arabidopsis Plants by Over-Expressingthe Genes PpPK-1, PpPK-2 and PpMPK-1

[0147] Binary Vector Construction: pGMSG

[0148] The pLMNC53 (Mankin, 2000, PhD thesis) vector was digested withHindIII (Roche) and blunt-end filled with Klenow enzyme and 0.1 mM dNTPs(Roche) according to manufacturer's instructions. This fragment wasextracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen)according to manufacturer's instructions. The purified fragment was thendigested with EcoRI (Roche) according to manufacturer's instructions.This fragment was extracted from agarose gel with a QIAquick GelExtraction Kit (Qiagen) according to manufacturer's instructions. Theresulting 1.4 kilobase fragment, the gentamycin cassette, included thenos promoter, aacCI gene and the g7 terminator.

[0149] The vector pBlueScript was digested with EcoRI and SmaI (Roche)according to manufacturer's instructions. The resulting fragment wasextracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen)according to manufacturer's instructions. The digested pBlueScriptvector and the gentamycin cassette fragments were ligated with T4 DNALigase (Roche) according to manufacturer's instructions, joining the tworespective EcoRI sites and joining the blunt-ended HindIII site with theSmaI site.

[0150] The recombinant vector (pGMBS) was transformed into Top10 cells(Invitrogen) using standard conditions. Transformed cells were selectedfor on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside), grown overnight at 37° C. Whitecolonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping was performed according to standard molecularbiology techniques (Sambrook et al. 1989. Molecular Cloning, ALaboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press.Cold Spring Harbor, N.Y.).

[0151] Both the pGMBS vector and p1bxSuperGUS vector were digested withXbaI and KpnI (Roche) according to manufacturer's instructions, excisingthe gentamycin cassette from pGMBS and producing the backbone from thep1bxSuperGUS vector. The resulting fragments were extracted from agarosegel with a QIAquick Gel Extraction Kit (Qiagen) according tomanufacturer's instructions. These two fragments were ligated with T4DNA ligase (Roche) according to manufacturer's instructions.

[0152] The resulting recombinant vector (pGMSG) was transformed intoTop10 cells (Invitrogen) using standard conditions. Transformed cellswere selected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mgX-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside) and grown overnight at 37° C. Whitecolonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping was performed according to standard molecularbiology techniques (Sambrook et al. 1989. Molecular Cloning, ALaboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press.Cold Spring Harbor, N.Y.).

[0153] Subcloning of PpPK-1, PpPK-2 and PpMPK-1 into the Binary Vector

[0154] The fragments containing the different Physcomitrella patensprotein kinase-like genes were subcloned from the recombinant PCR2.1TOPO vectors by double digestion with restriction enzymes (see Table 6)according to manufacturer's instructions. The subsequence fragment wasexcised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen)according to manufacturer's instructions and ligated into the binaryvector pGMSG, cleaved with appropriate enzymes (see Table 6) anddephosphorylated prior to ligation. The resulting recombinant pGMSGvector contained the corresponding transcription factor in the senseorientation under the control of the constitutive super promoter. TABLE6 Names of the various constructs of the Physcomitrella patens proteinkinase-like proteins used for plant transformation Enzymes used togenerate gene Enzymes used to Binary Vector Gene fragment restrict pGMSGConstruct PpPK-1 XmaI/SacI XmaI/SacI PBPSLVM004 PpPK-2 XmaI/HpaIXmaI/Ec1136 pBPSLVM005 PpMPK-1 XmaI/HpaI XmaI/Ec1136 PBPSLVM006

[0155] Agrobacterium Transformation

[0156] The recombinant vectors were transformed into Agrobacteriumtumefaciens C58C1 and PMP90 according to standard conditions (Hoefgenand Willmitzer, 1990).

[0157] Plant Transformation

[0158]Arabidopsis thaliana ecotype C24 were grown and transformedaccording to standard conditions (Bechtold 1993, Acad. Sci. Paris.316:1194-1199; Bent et al. 1994, Science 265:1856-1860).

[0159] Screening of Transformed Plants

[0160] T1 seeds were sterilized according to standard protocols (Xionget al. 1999, Plant Molecular Biology Reporter 17: 159-170). Seeds wereplated on ½ MS 0.6% agar supplemented with 1% sucrose, 150 μg/mlgentamycin (Sigma-Aldrich) and 2 μg/ml benomyl (Sigma-Aldrich). Seeds onplates were vernalized for four days at 4° C. The seeds were germinatedin a climatic chamber at an air temperature of 22° C. and lightintensity of 40 micromols^(−1m2) (white light; Philips TL 65W/25fluorescent tube) and 16 hours light and 8 hours dark day length cycle.Transformed seedlings were selected after 14 days and transferred to ½MS 0.6% agar plates supplemented with 1% sucrose and allowed to recoverfor five-seven days.

[0161] Drought Tolerance Screening

[0162] T1 seedlings were transferred to dry, sterile filter paper in apetri dish and allowed to desiccate for two hours at 80% RH (relativehumidity) in a Sanyo Growth Cabinet MLR-350H, micromols^(−1m2) (whitelight; Philips TL 65W/25 fluorescent tube). The RH was then decreased to60% and the seedlings were desiccated further for eight hours. Seedlingswere then removed and placed on ½ MS 0.6% agar plates supplemented with2 μg/ml benomyl and scored after five days.

[0163] The results of the drought tolerance screening in Arabidopsisthaliana plants over-expressing the Protein Kinase-like proteins areshown in Table 7. It is noteworthy that these analyses were performedwith T1 plants since the results should be better when a homozygous,strong expresser is found. TABLE 7 Summary of the drought stress testsDrought Stress Test Total number of Percentage of Gene Name Number ofsurvivors plants survivors PpPK-1 2 4 50% PpPK-2 10 12 84% PpMPK-1 23 2592% Control 18 84 21%

[0164] Salt Tolerance Screening

[0165] Seedlings were transferred to filter paper soaked in ½ MS andplaced on ½ MS 0.6% agar supplemented with 2 μg/ml benomyl the nightbefore the salt tolerance screening. For the salt tolerance screening,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked in 50 mM NaCl, in a petri dish. After two hours,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked with 200 mM NaCl, in a petri dish. After two hours,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked in 600 mM NaCl, in a petri dish. After 10 hours,the seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2 [Lg/ml benomyl. The seedlings were scored after 5days.

[0166] The results of the salt tolerance screening in Arabidopsisthaliana plants over-expressing the Protein Kinase-like proteins areshown in Table 8. It is noteworthy that these analyses were performedwith T1 plants since the results should be better when a homozygous,strong expresser is found. TABLE 8 Summary of the salt stress tests SaltStress Test Total number of Percentage of Gene Name Number of survivorsplants survivors PpPK-2 5 8 62% PpMPK-1 14 25 56% Control 4 43  9%

[0167] Freezing Tolerance Screening

[0168] Seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2% sucrose and 2 μg/ml benomyl. After four days, theseedlings were incubated at 4° C. for 1 hour and then covered withshaved ice. The seedlings were then placed in an EnvironmentalSpecialist ES2000 Environmental Chamber and incubated for 3.5 hoursbeginning at −1.0° C. decreasing −1° C. hour. The seedlings were thenincubated at −5.0° C. for 24 hours and then allowed to thaw at 5° C. for12 hours. The water was poured off and the seedlings were scored after 5days. The transgenic plants are then screened for their improved coldtolerance demonstrating that transgene expression confers coldtolerance.

Example 8

[0169] Detection of the PpPK-1, PpPK-2 and PpMPK-1 Transgenes in theTransgenic Arabidopsis Lines

[0170] One leaf from a wild type and a transgenic Arabidopsis plant washomogenized in 250 μl Hexadecyltrimethyl ammonium bromide (CTAB) buffer(2% CTAB, 1.4 M NaCl, 8mM EDTA and 20 mM Tris pH 8.0) and 1 μlβ-mercaptoethanol. The samples were incubated at 60-65° C. for 30minutes and 250 μl of Chloroform was then added to each sample. Thesamples were vortexed for 3 minutes and centrifuged for 5 minutes at18,000×g. The supernatant was taken from each sample and 150 μlisopropanol was added. The samples were incubated at room temperaturefor 15 minutes, and centrifuged for 10 minutes at 18,000×g. Each pelletwas washed with 70% ethanol, dried, and resuspended in 20 μl TE. 4 μl ofabove suspension was used in a 20 μl PCR reaction using Taq DNApolymerase (Roche Molecular Biochemicals) according to themanufacturer's instructions. Binary vector plasmid containing each STAgene was used as positive control, and the wild type C24 genomic DNA wasused as negative control in the PCR reactions. 10 μl PCR reaction wasanalyzed on 0.8% agarose-ethidium bromide gel.

[0171] Notably, the transgenes were successfully amplified from the T1transgenic lines, but not from the wild-type C24. This result indicatesthat the T1 transgenic plants contain at least one copy of thetransgenes. There was no indication of existence of either identical orvery similar in the untransformed Arabidopsis thaliana control which canbe amplified by this method.

[0172] PpPk-1

[0173] The primers used in the reactions were: SEQ ID NO:215′CTAGTAACATAGATGACACC3′ SEQ ID NO:225′ ATCCCGGGCGTTCAAGCAGGTGAATATGACAAC 3′

[0174] The PCR program was as following: 30 cycles of 1 minute at 94°C., 1 minute at 62° C. and 4 minutes at 72° C., followed by 10 minutesat 72° C. A 1.6 kb fragment was produced from the positive control andthe transgenic plants.

[0175] PpPk-2

[0176] The primers used in the reactions were: SEQ ID NO:235′GAATAGATACGCTGACACGC3′ SEQ ID NO:245′GCGTTAACGCCCGCAAAGGTCAAAACAGGCGTGG3′

[0177] The primers were used in the first round of reactions with thefollowing program: 30 cycles of 1 minute at 94° C., 1 minute at 62° C.and 4 minutes at 72° C., followed by 10 minutes at 72° C. Then 1 μl ofabove reaction was reamplified in a 20 μl reaction using the followingprimers in the same program: SEQ ID NO:255′ATCCCGGGCGCGCACAATTTCAGTTGGGAATCA3′ SEQ ID NO:245′GCGTTAACGCCCGCAAAGGTCAAAACAGGCGTGG3′

[0178] A 2.0 kb fragment was generated from the positive control and theT1 transgenic plants.

[0179] PpMPK-1

[0180] The primers used in the reactions were: SEQ ID NO:265′ATCCCGGGCGGTTTGGACACGATGTGTTCCAGTCC3′ SEQ ID NO:275′GCGTTAACTAACCGCGTTTAAGTCCCTCAAC3′

[0181] The PCR program was as following: 30 cycles of 1 minute at 94°C., 1 minute at 62° C. and 4 minutes at 72° C., followed by 10 minutesat 72° C. A 1.6 kb fragment was produced from the positive control andthe transgenic plants.

Example 9

[0182] Detection of the PpPK-1, PpPK-2 and PpMPK-1 Transgene mRNA inTransgenic Arabidopsis Lines

[0183] Transgene expression was detected using RT-PCR. Total RNA wasisolated from stress-treated plants using a procedure adapted from(Verwoerd et al. 1989. NAR 17:2362). Leaf samples (50-100 mg) werecollected and ground to a fine powder in liquid nitrogen. Ground tissuewas resuspended in 500 μl of a 80° C., 1:1 mixture, of phenol toextraction buffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS),followed by brief vortexing to mix. After the addition of 250 μl ofchloroform, each sample was vortexed briefly. Samples were thencentrifuged for 5 minutes at 12,000×g. The upper aqueous phase wasremoved to a fresh eppendorf tube. RNA was precipitated by adding{fraction (1/10)}^(th) volume 3M sodium acetate and 2 volumes 95%ethanol. Samples were mixed by inversion and placed on ice for 30minutes. RNA was pelleted by centrifugation at 12,000×g for 10 minutes.The supernatant was removed and pellets briefly air-dried. RNA samplepellets were resuspended in 10 μl DEPC treated water. To removecontaminating DNA from the samples, each was treated with RNase-freeDNase (Roche) according to the manufacturer's recommendations. cDNA wassynthesized from total RNA using the 1^(st) Strand cDNA synthesis kit(Boehringer Mannheim) following manufacturer's recommendations. PCRamplification of a gene-specific fragment from the synthesized cDNA wasperformed using Taq DNA polymerase (Roche) and gene-specific primers(see Table 5 for primers) in the following reaction: 1×PCR buffer, 1.5mM MgCl₂, 0.2 μM each primer, 0.2 μM dNTPs, 1 unit polymerase, 5 μl cDNAfrom synthesis reaction. Amplification was performed under the followingconditions: Denaturation, 95° C., 1 minute; annealing, 62° C., 30seconds; extension, 72° C., 1 minute, 35 cycles; extension, 72° C., 5minutes; hold, 4° C., forever. PCR products were run on a 1% agarosegel, stained with ethidium bromide, and visualized under UV light usingthe Quantity-One gel documentation system (Bio-Rad).

[0184] Expression of the transgenes was detected in the T1 transgenicline. These results indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic lines. In agreement with theprevious statement, no expression of identical or very similarendogenous genes could be detected by this method. These results are inagreement with the data from Example 7.

Example 10

[0185] Engineering Stress-Tolerant Soybean Plants by Over-Expressing thePpPK-1, PpPK-2 and PpMPK-1 Gene

[0186] The constructs pBPSLVM004, pBPSLVM005 and pBPSLVM006 were used totransform soybean as described below.

[0187] Seeds of soybean were surface sterilized with 70% ethanol for 4minutes at room temperature with continuous shaking, followed by 20%(v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes withcontinuous shaking. Then, the seeds were rinsed 4 times with distilledwater and placed on moistened sterile filter paper in a Petri dish atroom temperature for 6 to 39 hours. The seed coats were peeled off, andcotyledons are detached from the embryo axis. The embryo axis wasexamined to make sure that the meristematic region is not damaged. Theexcised embryo axes were collected in a half-open sterile Petri dish andair-dried to a moisture content less than 20% (fresh weight) in a sealedPetri dish until further use.

[0188]Agrobacterium tumefaciens culture was prepared from a singlecolony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/lstreptomycin, 50 mg/l kanamycin) followed by growth of the single colonyin liquid LB medium to an optical density at 600 nm of 0.8. Then, thebacteria culture was pelleted at 7000 rpm for 7 minutes at roomtemperature, and resuspended in MS (Murashige and Skoog, 1962) mediumsupplemented with 100 μM acetosyringone. Bacteria cultures wereincubated in this pre-induction medium for 2 hours at room temperaturebefore use. The axis of soybean zygotic seed embryos at approximately15% moisture content were imbibed for 2 hours at room temperature withthe pre-induced Agrobacterium suspension culture. The embryos areremoved from the imbibition culture and were transferred to Petri dishescontaining solid MS medium supplemented with 2% sucrose and incubatedfor 2 days, in the dark at room temperature. Alternatively, the embryoswere placed on top of moistened (liquid MS medium) sterile filter paperin a Petri dish and incubated under the same conditions described above.After this period, the embryos were transferred to either solid orliquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/Lcefotaxime to kill the agrobacteria. The liquid medium was used tomoisten the sterile filter paper. The embryos were incubated during 4weeks at 25° C., under 150 μmol m⁻²sec⁻¹ and 12 hours photoperiod. Oncethe seedlings produced roots, they were transferred to sterile metromixsoil. The medium of the in vitro plants was washed off beforetransferring the plants to soil. The plants were kept under a plasticcover for 1 week to favor the acclimatization process. Then the plantswere transferred to a growth room where they were incubated at 25° C.,under 150 μmol m⁻²sec⁻¹ light intensity and 12 hours photoperiod forabout 80 days.

[0189] The transgenic plants were then screened for their improveddrought, salt and/or cold tolerance according to the screening methoddescribed in Example 7 demonstrating that transgene expression confersstress tolerance.

Example 11

[0190] Engineering Stress-Tolerant Rapeseed/Canola Plants byOver-Expressing the PpPK-1, PpPK-2 and PpMPK-1 Gene

[0191] The constructs pBPSLVM004, pBPSLVM005 and pBPSLVM006 were used totransform rapeseed/canola as described below.

[0192] The method of plant transformation described herein is alsoapplicable to Brassica and other crops. Seeds of canola are surfacesterilized with 70% ethanol for 4 minutes at room temperature withcontinuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05%(v/v) Tween for 20 minutes, at room temperature with continuous shaking.Then, the seeds are rinsed 4 times with distilled water and placed onmoistened sterile filter paper in a Petri dish at room temperature for18 hours. Then the seed coats are removed and the seeds are air driedovernight in a half-open sterile Petri dish. During this period, theseeds lose approx. 85% of its water content. The seeds are then storedat room temperature in a sealed Petri dish until further use. DNAconstructs and embryo imbibition are as described in Example 10. Samplesof the primary transgenic plants (T0) are analyzed by PCR to confirm thepresence of T-DNA. These results are confirmed by Southern hybridizationin which DNA is electrophoresed on a 1% agarose gel and transferred to apositively charged nylon membrane (Roche Diagnostics). The PCR DIG ProbeSynthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

[0193] The transgenic plants are then screened for their improved stresstolerance according to the screening method described in Example 7demonstrating that transgene expression confers drought tolerance.

Example 12

[0194] Engineering Stress-Tolerant Corn Plants by Over-Expressing thePpCABF-1; PpDBF-1, PpCBF-1, PpHDZ-1, PpZF-1, PpLZ-1 and PpCABF-2 Gene

[0195] The constructs pBPSLVM004, pBPSLVM005 and pBPSLVM006 were used totransform corn as described below.

[0196] Transformation of maize (Zea Mays L.) is performed with themethod described by Ishida et al., 1996 Nature Biotch 14745-50. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. This procedure provides a transformation efficiency ofbetween 2.5% and 20%. The transgenic plants are then screened for theirimproved drought, salt and/or cold tolerance according to the screeningmethod described in Example 7 demonstrating that transgene expressionconfers stress tolerance.

Example 13

[0197] Engineering Stress-Tolerant Wheat Plants by Over-Expressing thePpPK-1, PpPK-2 and PpMPK-1 Gene

[0198] The constructs The constructs pBPSLVM004, pBPSLVM005 andpBPSLVM006 were used to transform wheat as described below.

[0199] Transformation of wheat is performed with the method described byIshida et al., 1996 Nature Biotch 14745-50. Immature embryos areco-cultivated with Agrobacterium tumefaciens that carry “super binary”vectors, and transgenic plants are recovered through organogenesis. Thisprocedure provides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved stress toleranceaccording to the screening method described in Example 7 demonstratingthat transgene expression confers drought tolerance.

Example 15

[0200] Identification of Homologous and Heterologous Genes

[0201] Gene sequences can be used to identify homologous or heterologousgenes from cDNA or genomic libraries. Homologous genes (e.g. full-lengthcDNA clones) can be isolated via nucleic acid hybridization using forexample EDNA libraries. Depending on the abundance of the gene ofinterest, 100,000 up to 1,000,000 recombinant bacteriophages are platedand transferred to nylon membranes. After denaturation with alkali, DNAis immobilized on the membrane by e.g. UV cross linking. Hybridizationis carried out at high stringency conditions. In aqueous solutionhybridization and washing is performed at an ionic strength of 1 M NaCland a temperature of 68° C. Hybridization probes are generated by e.g.radioactive (³²P) nick transcription labeling (High Prime, Roche,Mannheim, Germany). Signals are detected by autoradiography.

[0202] Partially homologous or heterologous genes that are related butnot identical can be identified in a manner analogous to theabove-described procedure using low stringency hybridization and washingconditions. For aqueous hybridization, the ionic strength is normallykept at 1 M NaCl while the temperature is progressively lowered from 68to 42° C.

[0203] Isolation of gene sequences with homologies (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radio labeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

[0204] Oligonucleotide hybridization solution:

[0205] 6×SSC

[0206] 0.01 M sodium phosphate

[0207] 1 mM EDTA (pH 8)

[0208] 0.5% SDS

[0209] 100 μg/ml denatured salmon sperm DNA

[0210] 0.1% nonfat dried milk

[0211] During hybridization, temperature is lowered stepwise to 5-10° C.below the estimated oligonucleotide Tm or down to room temperaturefollowed by washing steps and autoradiography. Washing is performed withlow stringency such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook, J. et al. (1989), “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.

Example 16

[0212] Identification of Homologous Genes by Screening ExpressionLibraries with Antibodies

[0213] c-DNA clones can be used to produce recombinant protein forexample in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinantproteins are then normally affinity purified via Ni—NTA affinitychromatography (Qiagen). Recombinant proteins are then used to producespecific antibodies for example by using standard techniques for rabbitimmunization. Antibodies are affinity purified using a Ni—NTA columnsaturated with the recombinant antigen as described by Gu et al., 1994BioTechniques 17:257-262. The antibody can than be used to screenexpression cDNA libraries to identify homologous or heterologous genesvia an immunological screening (Sambrook, J. et al. (1989), “MolecularCloning: A Laboratory Manual” , Cold Spring Harbor Laboratory Press orAusubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons).

Example 17

[0214] In vivo Mutagenesis

[0215] In vivo mutagenesis of microorganisms can be performed by passageof plasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2²9⁴, ASM: Washington.) Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M. (1994)Strategies 7: 32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 18

[0216] In vitro Analysis of the Function of Physcomitrella Genes inTransgenic Organisms

[0217] The determination of activities and kinetic parameters of enzymesis well established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M., and Webb, E. C.,(1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure andMechanism. Freeman: New York; Walsh, (1979) Enzymatic ReactionMechanisms. Freeman: San Francisco; Price, N. C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D.,ed. (1983) The Enzymes, 3^(rd) ed. Academic Press: New York; Bisswanger,H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim (ISBN 3527300325);Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds. (1983-1986) Methods ofEnzymatic Analysis, 3^(rd) ed., vol. I-XII, Verlag Chemie: Weinheim; andUllmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes.VCH: Weinheim, p. 352-363.

[0218] The activity of proteins which bind to DNA can be measured byseveral well-established methods, such as DNA band-shift assays (alsocalled gel retardation assays). The effect of such proteins on theexpression of other molecules can be measured using reporter gene assays(such as that described in Kolmar, H. et al. (1995) EMBO J. 14:3895-3904 and references cited therein). Reporter gene test systems arewell known and established for applications in both pro- and eukaryoticcells, using enzymes such as β-galactosidase, green fluorescent protein,and several others.

[0219] The determination of activity of membrane-transport proteins canbe performed according to techniques such as those described in Gennis,R. B. Pores, Channels and Transporters, in Biomembranes, MolecularStructure and Function, pp. 85-137, 199-234 and 270-322, Springer:Heidelberg (1989).

Example 19

[0220] Purification of the Desired Product from Transformed Organisms

[0221] Recovery of the desired product from plant material (i.e.,Physcomitrella patents or Arabidopsis thaliana), fungi, algae, ciliates,C. glutamicum cells, or other bacterial cells transformed with thenucleic acid sequences described herein, or the supernatant of theabove-described cultures can be performed by various methods well knownin the art. If the desired product is not secreted from the cells, canbe harvested from the culture by low-speed centrifugation, the cells canbe lysed by standard techniques, such as mechanical force orsonification. Organs of plants can be separated mechanically from othertissue or organs. Following homogenization cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from desired cells, then the cells areremoved from the culture by low-speed centrifugation, and the supernatefraction is retained for further purification.

[0222] The supernatant fraction from either purification method issubjected to chromatography with a suitable resin, in which the desiredmolecule is either retained on a chromatography resin while many of theimpurities in the sample are not, or where the impurities are retainedby the resin while the sample is not. Such chromatography steps may berepeated as necessary, using the same or different chromatographyresins. One skilled in the art would be Well-versed in the selection ofappropriate chromatography resins and in their most efficaciousapplication for a particular molecule to be purified. The purifiedproduct may be concentrated by filtration or ultrafiltration, and storedat a temperature at which the stability of the product is maximized.

[0223] There is a wide array of purification methods known to the artand the preceding method of purification is not meant to be limiting.Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: NewYork (1986). Additionally, the identity and purity of the isolatedcompounds may be assessed by techniques standard in the art. Theseinclude high-performance liquid chromatography (HPLC), spectroscopicmethods, staining methods, thin layer chromatography, NIRS, enzymaticassay, or microbiologically. Such analysis methods are reviewed in:Patek et al., 1994 Appl. Environ. Microbiol. 60:133-140; Malakhova etal., 1996 Biotekhnologiya 11:27-32; and Schmidt et al., 1998 BioprocessEngineer. 19:67-70. Ulmann's Encyclopedia of Industrial Chemistry,(1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p.559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons;Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.

1 28 1 326 DNA Physcomitrella patens 1 gcacgaggac aagggctact ctggggctactgccgatttg tggtcatgcg gtgttatttt 60 atatgtactg atggctggat acttgccatttgaggagccc acaatcatgg cactttacaa 120 gaagatttac cgagcacagt tctcttggcctccttggttc tcgtcaggag cccggaaatt 180 aatctcaaag atactggatc ccaatccaagaactcgcatt tctgcatctc aaatttataa 240 aaatgattgg tttaagaaag gatatactccagcccagttt gacagagaag ttgatgtcaa 300 ccttgacgat gtgaatgcta tcttta 326 2468 DNA Physcomitrella patens modified_base (1)..(468) “n” represents a,t, c, g, other or unknown 2 gcaccaggtc ctctacatgc ttatttcaac cctaaacctgaagggtcatt attatcagga 60 agtatggcaa gctctacaac gatttcttat ggatctagcatggccaacta cacatgcact 120 gctaagacgt tcactttagc agaattagaa agagcaacagataattttag acctgataat 180 gtagtcggcg aagggggctt tggtcgagtt taccaaggggtacttgatag tggtattgaa 240 gtggctgtaa aggtgctgac aagggatgat catgagggtggtcgtgagtt tgttgcggag 300 gtanaaatgt taagcagatt gcaccatcgc aaccttgtgaagctaattgg catctgtact 360 gaggaaatac gatgtttggt ttatgagctc atcacgaacgggagcgtgga atctcacttg 420 catggtctag ataaatacac tgctcctctc aactgggatgcacgtgtt 468 3 561 DNA Physcomitrella patens 3 gcacgaggtg acctgaagcccagtaatctc ctcatcaatt gcaacgactg tctactcaag 60 atttgtgatt ttggcttggctcgaacatct gcagaggatg acttccttac ggagtatgtt 120 gttactcgac catatcgagctccagagctc ttgcttggga gccgaatgta cacagcggct 180 gttgatatgt ggtcagtgggctgcatcttc atggagatgc ttacaggaca acctttgttt 240 ccaatccggt caaggcaagagcatcccgtg aatcatttga aactcatcac ggagcttcta 300 ggaacacccg atgcttcggacctgtcgttt ctgcagaatc cagatgctcg gcaaagaatc 360 caaatggctt tgttaggtcaggaaaggaag cctttgtttt cgaggtttcc tcaaacgtct 420 gcaatagctt gtgacttagcggagaagatg ctgaggttta acccatccaa cagaataact 480 gcggaagagg cttggcccatccttacttgg gcagcgcttc acgacctaag tgatgagcca 540 acgtgtcatc ttatgttcga c561 4 1424 DNA Physcomitrella patens 4 atcccgggcg ttcaagcagg tgaatatgacaactaaatca aacatgccga ctactaacgt 60 cgaacgtacg cgggtcggaa agtatgacctcggcaagacc ttgggagagg gtacatttgc 120 caaagtcaag gtggccaaac atattgacactggtcacact gttgccatca agattttgga 180 caaggagaag attctcaggc acaagatggtggaacagatc aaaagagaaa tatctaccat 240 gaagctggtg aaacatccct atgtcgtccagctgttggag gtaatggcca gcaggacgaa 300 gatctacatt gtgcttgagt atgtcacaggtggcgagctt tttaataaga ttgctcaaca 360 aggaagactg tcagaggatg aagcaaggaagtattttcag cagctgattg atgcagttga 420 ttattgccac agtcggcaag tgtatcacagagatctgaaa ccagagaatc ttcttctgga 480 ttccaaaggc aacttaaaaa tttccgactttggcttgagt gcgctacctc agcaatttag 540 ggaagatggt ttattacata caacttgcggaactcccaac tacgtggccc ctgaggttat 600 catggacaag ggctactctg gggctactgccgatttgtgg tcatgcggtg ttattttata 660 tgtactgatg gctggatact tgccatttgaggagcccaca atcatggcac tttacaagaa 720 gatttaccga gcacagttct cttggcctccttggttctcg tcaggagccc ggaaattaat 780 ctcaaagata ctggatccca atccaagaactcgcatttct gcatctcaaa tttataaaaa 840 tgattggttt aagaaaggat atactccagcccagtttgac agagaagttg atgtcaacct 900 tgacgatgtg aatgctatct ttagttgctcacaggaacat atggttgtcg aaaggaagga 960 aacaaagccg gtgtcaatga atgctttcgagcttatctcc atgtcatcag ggctcaacct 1020 ctccagcctc ttcgagacga aagagattcctgaaaaggag gacactaggt ttacgagtaa 1080 gaagtctgcg aaggagatca tctcttcaattgaggaagct gcaaagccct tagggttcaa 1140 cgttttgaaa cgtgatttca agctgaaactacaaggtcag ctggggagga agggacctct 1200 gtcagtttca actgaggtgt ttgaggtggcaccttctctt tacatggttg agttacagaa 1260 gaacagcggc gatacgttgg agtacaataacttttataag aatctttcca agggtctcaa 1320 agacatcgtg tggaaagcag accctattcctacaagtgag caaaagtaga aagcttccgc 1380 tacggcttta atatatgccc gtgctgtgaagtgcccgagc tcat 1424 5 2011 DNA Physcomitrella patens 5 atcccgggcgcgcacaattt cagttgggaa tcaagctgga aaagtttttt cctctagtgg 60 ctgagctggccaaggaactg gccattggac tcttcttaca aactagtcaa gtccgtattg 120 taggagccaatgctgttgaa cccaaccagg acaagacaaa cgtgagtgca gattttgtgc 180 cgctagataccaaatttgat cacaccactg cccatcttct tgctacacgc ttgtggagtg 240 gtgaagttccattgaacaag acactatttg gaacctacta tgttatttat ataatttacc 300 caggtcttcctccctctcca cctccccagt tccctgggaa tatttcacct tcaggtcctg 360 tcaaccagcttccatctggg gtggatccaa ataaaacaaa tcataaactc agttcgggaa 420 tgattaccgtgattgctttg gcttcggtta tgggtgtatt gttatttatt gggattgtat 480 ggctcattctcctacgccgc agcctggatg agaaaacttc gccttcggtt gtcggtcctc 540 tacatgcttatttcaaccct aaacctgaag gtgtgcaact gatccaactg agaatgaatg 600 cttatttcaactctaaacct gaagggtcat tattatcagg aagtatggca agctctacaa 660 cgatttcttatggatctagc atggccaact acacatgcac tgctaagacg ttcactttag 720 cagaattagaaagagcaaca gataatttta gacctgataa tgtagtcggc gaagggggct 780 ttggtcgagtttaccaaggg gtacttgata gtggtattga agtggctgta aaggtgctga 840 caagggatgatcatgagggt ggtcgtgagt ttgttgcgga ggtagaaatg ttaagcagat 900 tgcaccatcgcaaccttgcg aagctaattg gcatctgtac tgaggaaata cgatgtttgg 960 tttatgagctcatcacgaac gggagcgtgg aatctcactt gcatggtcta gataaataca 1020 ctgctcctctcaactgggat gcacgtgtta aaattgcatt aggagctgct cgtgggctgg 1080 catacctgcacgaagattct cagcctaggg ttattcatag agattttaaa ggaagcaaca 1140 ttctacttgaggacgattac actccaaaag tatctgattt tggtctagct aaatcggcaa 1200 ctgagggaggcaaggagcat atttccactc gagtaatggg cacgtttgga tatgtggctc 1260 ctgaatacgcaatgacagga catttgcttg tgaagagtga cgtttatagt tatggagtgg 1320 tactgctcgagctcctctcg gggcgtaaac ccgtggatat gtctcaacca cctggacaag 1380 agaatctagttacttgggca cgcccactcc ttacaagcaa ggatggacta gagcagcttg 1440 tggatccttacctcaaagac aactttccat ttgaccactt tgcgaaggta gctgcaatag 1500 cgtccatgtgtgtacaacct gaagtctctc atcgaccatt catgggcgag gtggtgcagg 1560 ccttgaaacttgtgtgcaat gaaacagaag ccaaagacgt cggacaggct aaaggaacag 1620 tttctcccacttctgacttg gccgaaacac agaacacagg atttctgcgg gacgccacct 1680 ttattagtgttgattacgac tcggggccct tcgaaacctt ggatcttgaa cagcgaaagc 1740 ggaaacctctttctgcttcg gctactatga gtggctctgg agggttctta cgacaacttt 1800 cggattcattcagacgctac agtgtttctg cccctccaaa ggctgcttca ctgccaagaa 1860 cttcatggtatgcactgggt agttcaaaac ctgtaggaag catgagtgag gctagagcag 1920 ctagattcttagatcctcaa cgcaggagat tttacgggtt ttggccctaa ttcttccacg 1980 cctgttttgacctttgcggg cgttaacgca a 2011 6 1627 DNA Physcomitrella patens 6atcccgggcg gtttggacac gatgttccag tcctttatat atatgagaca cagctggaga 60aaacagtgca agggaaaggt cttggaaagt ttttaatgca gttacttgag ttggttgcac 120gaaagaacaa catgaaagca gtacttttag ctgtgcataa aagaaacaca agggcgctaa 180ccttttacaa tgaacgttta gggtataagt tggcaattag atcagcatca agtcaacaaa 240gcacacaaac tgtcacagag atgaaatacg agattctttg taaaactttc gatgtggagt 300acacagccgt tgtagaggaa cggcaagggg acatggattg tgaatcacgt gaagagagcg 360ctggagaagc aagctgccag acagttgacg cagaggatca ggttttggat gactcacgtc 420ctgatacaga atgtgagtca cggatcgaga gcgtgccaaa caccctacaa ggaatgaagt 480acacacagta caatgtgagg ggcgacaagt ttgaagtcta cgacaagtat gtaatgattg 540gtcccattgg tcatggagct tatggcgatg tgtgtgcttt cacgaacagg gagacagggg 600agaaagtggc cataaagaag attggaaacg catttcagaa caatactaca gcgaggcgca 660cacttagaga gattttgttg ctccgccata ctgaacacga caacatcatt cccatcagag 720atatcattgt gcctgctaac attgaggact ttcatgatgc ctatatcgca aatgagctca 780tggatacaga ccttcaccag atagtgaggt caacaaaact tgacgaatac cattgccagt 840tcctgcttta ccagctgttg aggggtctca aatacatcca ctctgccaat atattgcacc 900gtgacctgaa gcccagtaat ctcctcatca attgcaacga ctgtctactc aagatttgtg 960attttggctt ggctcgaaca tctgcagagg atgacttcct tacggagtat gttgttactc 1020gaccatatcg agctccagag ctcttgcttg ggagccgaat gtacacagcg gctgttgata 1080tgtggtcagt gggctgcatc ttcatggaga tgcttacagg acaacctttg tttccaatcc 1140ggtcaaggca agggcatccc gtgaatcatt tgaaacccat cacggagctt ctaggaacac 1200ccgatgcttc ggacctgtcg tttctgcaga atccagatgc tcggcaaaga atccaaatgg 1260ctttgttagg tcaggaaagg aagcctttgt tttcgaggtt tcctcaaacg tctgcaatag 1320cttgtgactt agcggagaag atgctgaggt ttaacccatc caacagaata actgcggaag 1380aggccttggc ccatccttac ttggcagcgc ttcacgacct aagtgatgag ccaacgtgtc 1440atcttatgtt cgacttcgat gcttaccttc ccagcctaac agttgagcat gtgaaaactc 1500ttatctggag ggaagctaca cttatcaacg tccagtaatc gccataaaga tgtatcggac 1560cagatgtcgc tgcaccaatt ggcaaagctt aagggttgag ggacttaaac gcggttagtt 1620aacgcaa 1627 7 441 PRT Physcomitrella patens 7 Met Pro Thr Thr Asn ValGlu Arg Thr Arg Val Gly Lys Tyr Asp Leu 1 5 10 15 Gly Lys Thr Leu GlyGlu Gly Thr Phe Ala Lys Val Lys Val Ala Lys 20 25 30 His Ile Asp Thr GlyHis Thr Val Ala Ile Lys Ile Leu Asp Lys Glu 35 40 45 Lys Ile Leu Arg HisLys Met Val Glu Gln Ile Lys Arg Glu Ile Ser 50 55 60 Thr Met Lys Leu ValLys His Pro Tyr Val Val Gln Leu Leu Glu Val 65 70 75 80 Met Ala Ser ArgThr Lys Ile Tyr Ile Val Leu Glu Tyr Val Thr Gly 85 90 95 Gly Glu Leu PheAsn Lys Ile Ala Gln Gln Gly Arg Leu Ser Glu Asp 100 105 110 Glu Ala ArgLys Tyr Phe Gln Gln Leu Ile Asp Ala Val Asp Tyr Cys 115 120 125 His SerArg Gln Val Tyr His Arg Asp Leu Lys Pro Glu Asn Leu Leu 130 135 140 LeuAsp Ser Lys Gly Asn Leu Lys Ile Ser Asp Phe Gly Leu Ser Ala 145 150 155160 Leu Pro Gln Gln Phe Arg Glu Asp Gly Leu Leu His Thr Thr Cys Gly 165170 175 Thr Pro Asn Tyr Val Ala Pro Glu Val Ile Met Asp Lys Gly Tyr Ser180 185 190 Gly Ala Thr Ala Asp Leu Trp Ser Cys Gly Val Ile Leu Tyr ValLeu 195 200 205 Met Ala Gly Tyr Leu Pro Phe Glu Glu Pro Thr Ile Met AlaLeu Tyr 210 215 220 Lys Lys Ile Tyr Arg Ala Gln Phe Ser Trp Pro Pro TrpPhe Ser Ser 225 230 235 240 Gly Ala Arg Lys Leu Ile Ser Lys Ile Leu AspPro Asn Pro Arg Thr 245 250 255 Arg Ile Ser Ala Ser Gln Ile Tyr Lys AsnAsp Trp Phe Lys Lys Gly 260 265 270 Tyr Thr Pro Ala Gln Phe Asp Arg GluVal Asp Val Asn Leu Asp Asp 275 280 285 Val Asn Ala Ile Phe Ser Cys SerGln Glu His Met Val Val Glu Arg 290 295 300 Lys Glu Thr Lys Pro Val SerMet Asn Ala Phe Glu Leu Ile Ser Met 305 310 315 320 Ser Ser Gly Leu AsnLeu Ser Ser Leu Phe Glu Thr Lys Glu Ile Pro 325 330 335 Glu Lys Glu AspThr Arg Phe Thr Ser Lys Lys Ser Ala Lys Glu Ile 340 345 350 Ile Ser SerIle Glu Glu Ala Ala Lys Pro Leu Gly Phe Asn Val Leu 355 360 365 Lys ArgAsp Phe Lys Leu Lys Leu Gln Gly Gln Leu Gly Arg Lys Gly 370 375 380 ProLeu Ser Val Ser Thr Glu Val Phe Glu Val Ala Pro Ser Leu Tyr 385 390 395400 Met Val Glu Leu Gln Lys Asn Ser Gly Asp Thr Leu Glu Tyr Asn Asn 405410 415 Phe Tyr Lys Asn Leu Ser Lys Gly Leu Lys Asp Ile Val Trp Lys Ala420 425 430 Asp Pro Ile Pro Thr Ser Glu Gln Lys 435 440 8 516 PRTPhyscomitrella patens 8 Met Ile Thr Val Ile Ala Leu Ala Ser Val Met GlyVal Leu Leu Phe 1 5 10 15 Ile Gly Ile Val Trp Leu Ile Leu Leu Arg ArgSer Leu Asp Glu Lys 20 25 30 Thr Ser Pro Ser Val Val Gly Pro Leu His AlaTyr Phe Asn Pro Lys 35 40 45 Pro Glu Gly Val Gln Leu Ile Gln Leu Arg MetAsn Ala Tyr Phe Asn 50 55 60 Ser Lys Pro Glu Gly Ser Leu Leu Ser Gly SerMet Ala Ser Ser Thr 65 70 75 80 Thr Ile Ser Tyr Gly Ser Ser Met Ala AsnTyr Thr Cys Thr Ala Lys 85 90 95 Thr Phe Thr Leu Ala Glu Leu Glu Arg AlaThr Asp Asn Phe Arg Pro 100 105 110 Asp Asn Val Val Gly Glu Gly Gly PheGly Arg Val Tyr Gln Gly Val 115 120 125 Leu Asp Ser Gly Ile Glu Val AlaVal Lys Val Leu Thr Arg Asp Asp 130 135 140 His Glu Gly Gly Arg Glu PheVal Ala Glu Val Glu Met Leu Ser Arg 145 150 155 160 Leu His His Arg AsnLeu Ala Lys Leu Ile Gly Ile Cys Thr Glu Glu 165 170 175 Ile Arg Cys LeuVal Tyr Glu Leu Ile Thr Asn Gly Ser Val Glu Ser 180 185 190 His Leu HisGly Leu Asp Lys Tyr Thr Ala Pro Leu Asn Trp Asp Ala 195 200 205 Arg ValLys Ile Ala Leu Gly Ala Ala Arg Gly Leu Ala Tyr Leu His 210 215 220 GluAsp Ser Gln Pro Arg Val Ile His Arg Asp Phe Lys Gly Ser Asn 225 230 235240 Ile Leu Leu Glu Asp Asp Tyr Thr Pro Lys Val Ser Asp Phe Gly Leu 245250 255 Ala Lys Ser Ala Thr Glu Gly Gly Lys Glu His Ile Ser Thr Arg Val260 265 270 Met Gly Thr Phe Gly Tyr Val Ala Pro Glu Tyr Ala Met Thr GlyHis 275 280 285 Leu Leu Val Lys Ser Asp Val Tyr Ser Tyr Gly Val Val LeuLeu Glu 290 295 300 Leu Leu Ser Gly Arg Lys Pro Val Asp Met Ser Gln ProPro Gly Gln 305 310 315 320 Glu Asn Leu Val Thr Trp Ala Arg Pro Leu LeuThr Ser Lys Asp Gly 325 330 335 Leu Glu Gln Leu Val Asp Pro Tyr Leu LysAsp Asn Phe Pro Phe Asp 340 345 350 His Phe Ala Lys Val Ala Ala Ile AlaSer Met Cys Val Gln Pro Glu 355 360 365 Val Ser His Arg Pro Phe Met GlyGlu Val Val Gln Ala Leu Lys Leu 370 375 380 Val Cys Asn Glu Thr Glu AlaLys Asp Val Gly Gln Ala Lys Gly Thr 385 390 395 400 Val Ser Pro Thr SerAsp Leu Ala Glu Thr Gln Asn Thr Gly Phe Leu 405 410 415 Arg Asp Ala ThrPhe Ile Ser Val Asp Tyr Asp Ser Gly Pro Phe Glu 420 425 430 Thr Leu AspLeu Glu Gln Arg Lys Arg Lys Pro Leu Ser Ala Ser Ala 435 440 445 Thr MetSer Gly Ser Gly Gly Phe Leu Arg Gln Leu Ser Asp Ser Phe 450 455 460 ArgArg Tyr Ser Val Ser Ala Pro Pro Lys Ala Ala Ser Leu Pro Arg 465 470 475480 Thr Ser Trp Tyr Ala Leu Gly Ser Ser Lys Pro Val Gly Ser Met Ser 485490 495 Glu Ala Arg Ala Ala Arg Phe Leu Asp Pro Gln Arg Arg Arg Phe Tyr500 505 510 Gly Phe Trp Pro 515 9 480 PRT Physcomitrella patens 9 MetGln Leu Leu Glu Leu Val Ala Arg Lys Asn Asn Met Lys Ala Val 1 5 10 15Leu Leu Ala Val His Lys Arg Asn Thr Arg Ala Leu Thr Phe Tyr Asn 20 25 30Glu Arg Leu Gly Tyr Lys Leu Ala Ile Arg Ser Ala Ser Ser Gln Gln 35 40 45Ser Thr Gln Thr Val Thr Glu Met Lys Tyr Glu Ile Leu Cys Lys Thr 50 55 60Phe Asp Val Glu Tyr Thr Ala Val Val Glu Glu Arg Gln Gly Asp Met 65 70 7580 Asp Cys Glu Ser Arg Glu Glu Ser Ala Gly Glu Ala Ser Cys Gln Thr 85 9095 Val Asp Ala Glu Asp Gln Val Leu Asp Asp Ser Arg Pro Asp Thr Glu 100105 110 Cys Glu Ser Arg Ile Glu Ser Val Pro Asn Thr Leu Gln Gly Met Lys115 120 125 Tyr Thr Gln Tyr Asn Val Arg Gly Asp Lys Phe Glu Val Tyr AspLys 130 135 140 Tyr Val Met Ile Gly Pro Ile Gly His Gly Ala Tyr Gly AspVal Cys 145 150 155 160 Ala Phe Thr Asn Arg Glu Thr Gly Glu Lys Val AlaIle Lys Lys Ile 165 170 175 Gly Asn Ala Phe Gln Asn Asn Thr Thr Ala ArgArg Thr Leu Arg Glu 180 185 190 Ile Leu Leu Leu Arg His Thr Glu His AspAsn Ile Ile Pro Ile Arg 195 200 205 Asp Ile Ile Val Pro Ala Asn Ile GluAsp Phe His Asp Ala Tyr Ile 210 215 220 Ala Asn Glu Leu Met Asp Thr AspLeu His Gln Ile Val Arg Ser Thr 225 230 235 240 Lys Leu Asp Glu Tyr HisCys Gln Phe Leu Leu Tyr Gln Leu Leu Arg 245 250 255 Gly Leu Lys Tyr IleHis Ser Ala Asn Ile Leu His Arg Asp Leu Lys 260 265 270 Pro Ser Asn LeuLeu Ile Asn Cys Asn Asp Cys Leu Leu Lys Ile Cys 275 280 285 Asp Phe GlyLeu Ala Arg Thr Ser Ala Glu Asp Asp Phe Leu Thr Glu 290 295 300 Tyr ValVal Thr Arg Pro Tyr Arg Ala Pro Glu Leu Leu Leu Gly Ser 305 310 315 320Arg Met Tyr Thr Ala Ala Val Asp Met Trp Ser Val Gly Cys Ile Phe 325 330335 Met Glu Met Leu Thr Gly Gln Pro Leu Phe Pro Ile Arg Ser Arg Gln 340345 350 Gly His Pro Val Asn His Leu Lys Pro Ile Thr Glu Leu Leu Gly Thr355 360 365 Pro Asp Ala Ser Asp Leu Ser Phe Leu Gln Asn Pro Asp Ala ArgGln 370 375 380 Arg Ile Gln Met Ala Leu Leu Gly Gln Glu Arg Lys Pro LeuPhe Ser 385 390 395 400 Arg Phe Pro Gln Thr Ser Ala Ile Ala Cys Asp LeuAla Glu Lys Met 405 410 415 Leu Arg Phe Asn Pro Ser Asn Arg Ile Thr AlaGlu Glu Ala Leu Ala 420 425 430 His Pro Tyr Leu Ala Ala Leu His Asp LeuSer Asp Glu Pro Thr Cys 435 440 445 His Leu Met Phe Asp Phe Asp Ala TyrLeu Pro Ser Leu Thr Val Glu 450 455 460 His Val Lys Thr Leu Ile Trp ArgGlu Ala Thr Leu Ile Asn Val Gln 465 470 475 480 10 18 DNA ArtificialSequence Description of Artificial Sequence Primer 10 caggaaacagctatgacc 18 11 19 DNA Artificial Sequence Description of ArtificialSequence Primer 11 ctaaagggaa caaaagctg 19 12 18 DNA Artificial SequenceDescription of Artificial Sequence Primer 12 tgtaaaacga cggccagt 18 1325 DNA Artificial Sequence Description of Artificial Sequence Primer 13ccgggctcct gacgagaacc aagga 25 14 33 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 14 atcccgggcg ttcaagcagg tgaatatgac aac 3315 32 DNA Artificial Sequence Description of Artificial Sequence Primer15 atgagctcgg gcacttcaca gcacgggcat at 32 16 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 16 gcccccttcg ccgactacat tatca25 17 33 DNA Artificial Sequence Description of Artificial SequencePrimer 17 atcccgggcg cgcacaattt cagttgggaa tca 33 18 34 DNA ArtificialSequence Description of Artificial Sequence Primer 18 gcgttaacgcccgcaaaggt caaaacaggc gtgg 34 19 26 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 19 gccgctgtgt acattcggct cccaag 26 20 33DNA Artificial Sequence Description of Artificial Sequence Primer 20atcccgggcg gtttggacac gatgttccag tcc 33 21 31 DNA Artificial SequenceDescription of Artificial Sequence Primer 21 gcgttaacta accgcgtttaagtccctcaa c 31 22 20 DNA Artificial Sequence Description of ArtificialSequence Primer 22 ctagtaacat agatgacacc 20 23 33 DNA ArtificialSequence Description of Artificial Sequence Primer 23 atcccgggcgttcaagcagg tgaatatgac aac 33 24 20 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 24 gaatagatac gctgacacgc 20 25 34 DNAArtificial Sequence Description of Artificial Sequence Primer 25gcgttaacgc ccgcaaaggt caaaacaggc gtgg 34 26 33 DNA Artificial SequenceDescription of Artificial Sequence Primer 26 atcccgggcg cgcacaatttcagttgggaa tca 33 27 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 27 atcccgggcg gtttggacac gatgttccag tcc 33 2831 DNA Artificial Sequence Description of Artificial Sequence Primer 28gcgttaacta accgcgttta agtccctcaa c 31

We claim:
 1. A transgenic plant transformed by a protein kinasestress-related protein (PKSRP) coding nucleic acid, wherein expressionof the nucleic acid sequence in the plant results in increased toleranceto environmental stress as compared to a wild type variety of the plant.2. The transgenic plant of claim 1, wherein the PKSRP is selectedfrom 1) Receptor Protein Kinases (RPK); 2) Receptor-Like Kinases (RLK);3) Calcium Dependent Protein Kinases (CDPK); 4) SNF1 Serine/threonineProtein Kinases (SNF1); 5) Mitogen-activated Protein Kinases (MAPK); 6)intermediate upstream Mitogen-activated Protein Kinases (MAPKK); andupstream Mitogen-activated Protein Kinases (MAPKKK).
 3. The transgenicplant of claim 1, wherein the PKSRP is selected from 1) Protein Kinase-1(PK-1) as defined in SEQ ID NO: 7; 2) Protein Kinase-1 (PK-2) as definedin SEQ ID NO: 8; and 3) Mitogen-activated Protein Kinase-1 (MPK-1) asdefined in SEQ ID NO: 9; and homologues thereof.
 4. The transgenic plantof claim 1, wherein the PKSRP coding nucleic acid is selected from 1)Protein Kinase-1 (PK-1) as defined in SEQ ID NO: 4; 2) Protein Kinase-1(PK-2) as defined in SEQ ID NO: 5; and 3) Mitogen-activated ProteinKinase-1 (MPK-1) as defined in SEQ ID NO: 6; and homologues thereof. 5.The transgenic plant of any of claims 3 or 4, wherein the nucleic acidand protein are from a Physcomitrella patens.
 6. The transgenic plant ofclaim 1, wherein the PKSRP is selected from 1) Protein Kinase-1 (PK-1)as defined in SEQ ID NO: 7; 2) Protein Kinase-1 (PK-2) as defined in SEQID NO: 8; and 3) Mitogen-activated Protein Kinase-1 (MPK-1) as definedin SEQ ID NO:
 9. 7. The transgenic plant of claim 1, wherein the PKSRPcoding nucleic acid is selected from 1) Protein Kinase-1 (PK-1) asdefined in SEQ ID NO: 4; 2) Protein Kinase-1 (PK-2) as defined in SEQ IDNO: 5; and 3) Mitogen-activated Protein Kinase-1 (MPK-1) as defined inSEQ ID NO:
 6. 8. The transgenic plant of claim 1, wherein theenvironmental stress is selected from salinity, drought, andtemperature.
 9. A transgenic plant of any of claims 2, 3, 5 and 6,wherein the environmental stress is salinity, and the PKSRP is selectedfrom PK-2 and MPK-1.
 10. A transgenic plant of any of claims 4, 5 and 7,wherein the environmental stress is salinity, and the PKSRP codingnucleic acid is selected from PK-2 and MPK-1.
 11. A transgenic plant ofany of claims 2, 3, 5 and 6, wherein the environmental stress isdrought, and the PKSRP is selected from PK-1, PK-2 and MPK-1.
 12. Atransgenic plant of any of claims 4, 5 and 7, wherein the environmentalstress is drought, and the PKSRP coding nucleic acid is selected fromPK-1, PK-2 and MPK-1. The transgenic plant of any of claims 1-12,wherein the plant is a monocot. The transgenic plant of any of claims1-12, wherein the plant is a dicot.
 15. The transgenic plant of any ofclaims 1-12, wherein the plant is selected from maize, wheat, rye, oat,triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola,manihot, pepper, sunflower, tagetes, solanaceous plants, potato,tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,tea, Salix species, oil palm, coconut, perennial grass and forage crops.16. A seed produced by a transgenic plant transformed by a proteinkinase stress-related protein (PKSRP) coding nucleic acid, wherein theseed contains the PKSRP of any of claims 2, 3, 5, 6, 8, 9 and 11, andwherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.17. A seed produced by a transgenic plant transformed by a proteinkinase stress-related protein (PKSRP) coding nucleic acid, wherein theseed contains the PKSRP coding nucleic acid of any of claims 4, 5, 7, 8,10 and 12, and wherein the plant is true breeding for increasedtolerance to environmental stress as compared to a wild type variety ofthe plant.
 18. An agricultural product produced by the plant or seed ofany of claims 1-17.
 19. An isolated protein kinase stress-relatedprotein (PKSRP), wherein the PKSRP is as described in any of claims 2,3, 5, 6, 8, 9 and
 11. 20. An isolated protein kinase stress-relatedprotein (PKSRP) coding nucleic acid, wherein the PKSRP coding nucleicacid codes for a PKSRP as described in any of claims 2, 3, 5, 6, 8, 9and
 11. 21. An isolated protein kinase stress-related protein (PKSRP)coding nucleic acid, wherein the PKSRP coding nucleic acid is asdescribed in any of claims 4, 5, 7, 8, 10 and
 12. 22. An isolatedrecombinant expression vector comprising a nucleic acid of any of claims20 and 21, wherein expression of the vector in a host cell results inincreased tolerance to environmental stress as compared to a wild typevariety of the host cell.
 23. A host cell containing the vector of claim22.
 24. The host cell of claim 23, wherein the cell is in a plant.
 25. Amethod of producing a transgenic plant with a protein kinasestress-related protein (PKSRP) coding nucleic acid, wherein expressionof the nucleic acid in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising, (a) transforming a plant cell with an expression vectorcomprising the PKSRP coding nucleic acid, and (b) generating from theplant cell a transgenic plant with an increased tolerance toenvironmental stress as compared to a wild type variety of the plant.26. The method of claim 25, wherein the PKSRP is as described in any ofclaims 2, 3, 5, 6, 8, 9 and
 11. 27. The method of claim 25, wherein thePKSRP coding nucleic acid is as described in any of claims 4, 5, 7, 8,10 and
 12. 28. A method of identifying a novel protein kinasestress-related protein (PKSRP) comprising, (a) raising a specificantibody response to a PKSRP as described in any of claims 2, 3, 5, 6,8, 9 and 11; (b) screening putative PKSRP material with the antibody,wherein specific binding of the antibody to the material indicates thepresence of a potentially novel PKSRP; and (c) identifying the boundmaterial as a novel PKSRP.
 29. A method of modifying stress tolerance ofa plant comprising, modifying the expression of a protein kinasestress-related protein (PKSRP) in the plant, wherein the PKSRP is asdescribed in any of claims 2, 3, 5, 6, 8, 9 and
 11. 30. The method ofclaim 29, wherein the stress tolerance is increased.
 31. The method ofclaim 29, wherein the stress tolerance is decreased.
 32. The method ofclaim 29, wherein the plant is not transgenic.
 33. The method of claim29, wherein the plant is transgenic.
 34. The method of claim 33, whereinthe plant is transformed with PKSRP coding nucleic acid as described inany of claims 4, 5, 7, 8, 10 and
 12. 35. The method of claim 33, whereinthe plant is transformed with a promoter that directs expression ofnative PKSRP.
 36. The method of claim 35, wherein the promoter is tissuespecific.
 37. The method of claim 35, wherein the promoter isdevelopmentally regulated.
 38. The method of claim 29, wherein PKSRPexpression is modified by administration of an anti-sense molecule thatinhibits expression of PKSRP.